Initial Uptake Velocity Research Articles

Mechanism of isoniazid uptake in Mycobacterium tuberculosis.

Initial transport kinetics of isoniazid (INH) and its uptake at the plateau were studied in Mycobacterium tuberculosis H37Rv under various experimental conditions. The initial uptake velocity increased linearly with INH concentration from 2 x 10(-6) M to 10(-2) M. It was modified neither by addition of a protonophore that abolished proline transport, nor following ATP depletion by arsenate, which inhibited glycerol uptake, two transport processes taken as controls for secondary active transport and facilitated diffusion, respectively. Microaerobiosis or low temperature (4 degrees C) were without effect on initial uptake. It is thus likely that INH transport in M. tuberculosis proceeds by a passive diffusion mechanism, and that catalase-peroxidase (KatG) is not involved in the actual transport. However, conditions inhibiting KatG activity (high INH concentration, microaerobiosis, low temperature) decrease cell radioactivity at the uptake plateau. It is proposed that INH transport occurs by passive diffusion. KatG is involved only in the intracellular accumulation of oxidized derivatives of INH, especially of isonicotinic acid, which is trapped inside cells in its ionized form. This model explains observed and previously known characteristics of the accumulation of radioactivity in the presence of [14C]INH for various species and strains of mycobacteria.

Effect of amino acid ergot alkaloids on glutamate transport via human glutamate transporter hGluT-1

Effect of amino acid ergot alkaloids on glutamate transport via the human glutamate transporter (hGluT-1) was investigated using hGluT-HeLaS3 cells, which stably expressed high levels of hGluT-1. Ergotamine enhanced the glutamate uptake of hGluT-HeLaS3 cells in a concentration-dependent manner, increasing the initial velocity of glutamate uptake by 1.45 times at 10 μM. Other amino acid alkaloids, bromocriptine and dihydroergotamine, also augmented glutamate uptake, whereas amine alkaloids, ergonovine or lisuride did not influence glutamate uptake. The accelerating effect required a preincubation longer than 5 min. Kinetic studies on hGluT-1 revealed that ergot alkaloids decreased a Michaelis constant ( K m) for glutamate with unchanged maximum velocity. The effect of bromocriptine was not mediated by dopamine D 1 or D 2 receptors, and was independent of its antioxidant action. Amino acid ergot alkaloids may serve as a prototype for agents that regulate glutamate transporters. These results may be useful in exploring new agents for neurological diseases associated with glutamatergic neurotoxicity.

The Spermidine Transport System Is Regulated by Ligand Inactivation, Endocytosis, and by the Npr1p Ser/Thr Protein Kinase in Saccharomyces cerevisiae

We have characterized the regulation of spermidine transport in yeast and identified some of the genes involved in its control. Disruption of the SPE2 gene encoding S-adenosylmethionine decarboxylase, which catalyzes an essential step in polyamine biosynthesis, upregulated the initial velocity of spermidine uptake in wild-type cells as well as in the polyamine transport-deficient pcp1 mutants. Exogenous spermidine rapidly inactivated spermidine transport with a half-life of approximately 10-15 min via a process that did not require de novo protein synthesis but was accelerated by cycloheximide addition. Conversely, reactivation of spermidine influx upon polyamine deprivation required active protein synthesis. The stability of polyamine carrier activity was increased 2-fold in polyamine-depleted spe2 deletion mutants, indicating that endogenous polyamines also contribute to the down-regulation of spermidine transport. Ligand-mediated repression of spermidine transport was delayed in end3 and end4 mutants that are deficient in the initial steps of the endocytic pathway, and spermidine uptake activity was increased 4- to 5-fold in end3 mutants relative to parental cells, although the stability of the transport system was similar in both strains. Disruption of the NPR1 gene, which encodes a putative Ser/Thr protein kinase essential for the reactivation of several nitrogen permeases, resulted in a 3-fold decrease in spermidine transport in NH4(+)-rich media but did not prevent its down-regulation by spermidine. The defect in spermidine transport was more pronounced in NH4(+)- than proline-grown npr1 cells, suggesting that NPR1 protects against nitrogen catabolite repression of polyamine uptake activity. These results suggest that (a) the polyamine carrier is an unstable protein subject to down-regulation by spermidine via a process involving ligand inactivation followed by endocytosis and that (b) NPR1 expression fully prevents nitrogen catabolite repression of polyamine transport, unlike the role predicted for that gene by the inactivation/reactivation model proposed for other nitrogen permeases.

Methamphetamine-induced alterations in dopamine transporter function

Repeated methamphetamine (METH) administration has been shown to produce differing neurochemical as well as behavioral effects in rats. This study was designed to examine the effects of acute and chronic METH exposure on uptake and release of [ 3H]dopamine (DA) in cultured midbrain dopamine neurons to determine if persistent neuronal adaptations ensue. In addition, we have assessed DA D2 receptor function to determine if chronic METH alters this receptor. Fetal midbrain cultures were exposed to METH (1, 10 μM) for 5 days and dopaminergic function examined 1 or 7 days after drug removal. The ability of METH to release [ 3H]DA was compared to other releasing agents as well as several potent uptake inhibitors. Chronic exposure to a release-promoting concentration of METH resulted in either no change or a reduction in [ 3H]DA release upon subsequent METH challenge. Pretreatment with METH was also found to cause a decrease in the B max for [ 3H]raclopride binding, suggesting that persistently elevated DA levels cause a downregulation of DA D2 receptors. Examination of transporter kinetics utilizing initial velocity of uptake revealed that METH treatment caused a significant decrease in affinity ( K m) for the substrate (DA), while not altering the maximal velocity of uptake ( V max). Binding studies with [ 125I]RTI-55 revealed that there was no alteration in either the B max or K d for this ligand, suggesting that the changes induced by METH treatment are due to alterations in K m and not in the number of DA transport sites. The results from these studies indicate that METH treatment produces a modification in transporter function which may be associated with both the altered uptake and release of [ 3H]DA. These changes have broad implications for the regulation of transporter activity not only because of the relevance to pre-synaptic mechanisms controlling neurotransmission, but also to the importance of the neuronal adaptation that occurs in response to chronic METH exposure.

Regulation of the function of P-glycoprotein by epidermal growth factor through phospholipase C

Many multidrug-resistam (MDR) cell lines overexpress the epidermal growth factor receptor (EGFR) as well as P-glycoprotein (P-gp). However, the role of the increased EGFR in P-gp-mediated drug resistance remains unclear. Since recent studies suggest that activation of phospholipase C (PLC) could increase the phosphorylation of P-gp, and activation of the EGFR would also activate PLC, we investigated whether the effect of epidermal growth factor (EGF) on the phosphorylation of P-gp was mediated through PLC. Treatment of the human MDR hreast cancer cell line, MCF-7/AdrR, with EGF increased the phosphorylation of P-gp by 20–50%. The increased phosphorylation of P-gp was accompanied by stimulation of PLC activity, as measured by the production of inositol, 1,4,5-trisphosphate and diacylglycerol, products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Treatment of MDR cells with EGF also had detectable effects on P-gp function. For example, following incubation of MCF-7/AdrR cells with EGF, we observed a consistent decrease in total vinblastine (VBL) accumulation. Kinetic analysis revealed this change to be due to an increase in membrane efflux. The latter was measured by the initial uptake velocity, which was inhibited by EGF. VBL uptake measured at 0–320 sec was inhibited by 20–40%, which was associated with a similar increase in VBL efflux. EGF had no effect on drug accumulation, uptake, or efflux in sensitive MCF-7 cells. These data indicate that EGF can modulate the phosphorylation and function of P-gp, and suggest that this effect may be initiated by the activation of PLC.

Effect of surface and intracellular pH on hepatocellular fatty acid uptake

Fatty acids enter hepatocytes, at least in part, by a carrier-mediated uptake mechanism. The importance of driving forces for fatty acid uptake is still controversial. To evaluate possible driving mechanisms for fatty acid transport across plasma membranes, we examined the role of transmembrane proton gradients on fatty acid influx in primary cultured rat hepatocytes. After hepatocytes were loaded with SNARF-1 acetoxymethyl ester, changes in intracellular pH (pHi) under different experimental conditions were measured and recorded by confocal laser scanning microscopy. Fatty acid transport was increased by 45% during cellular alkalosis, achieved by adding 20 mM NH4Cl to the medium, and a concomitant paracellular acidification was observed. Fatty acid uptake was decreased by 30% during cellular acidosis after withdrawal of NH4Cl from the medium. Cellular acidosis activates the Na+/H+ antiporter to export excessive protons to the outer cell surface. Inhibition of Na+/H+ antiporter activity by amiloride diminishes pHi recovery and thereby accumulation of protons at the outer surface of the plasma membrane. Under these conditions, fatty acid uptake was further inhibited by 57% of control conditions. This suggests stimulation of fatty acid influx by an inwardly directed proton gradient. The accelerating effect of protons at the outer surface of the plasma membrane was confirmed by studies in which pH of the medium was varied at constant pHi. Significantly higher fatty acid influx rates were observed at low buffer pH. Recorded differences in fatty acid uptake appeared to be independent of changes in membrane potential, because BaCl2 did not influence initial uptake velocity during cellular alkalosis and paracellular acidosis. Moreover, addition of oleate-albumin mixtures to the NH4Cl incubation buffer did not change the observed intracellular alkalinization. In contrast, after cells were acid loaded, addition of oleate-albumin solutions to the recovery buffer increased pHi recovery rates from 0.21 +/- 0.02 to 0.36 +/- 0.05 pH units/min (P < 0.05), indicating that fatty acids further stimulate Na+/H+ antiporter activity during pHi recovery from an acid load. It is concluded that carrier-mediated uptake of fatty acids in hepatocytes follows an inwardly directed transmembrane proton gradient and is stimulated by the presence of H+ at the outer surface of the plasma membrane.

Relationship between Parathion and Paraoxon Toxicokinetics, Lung Metabolic Activity, and Cholinesterase Inhibition in Guinea Pig and Rabbit Lungs

Kinetic parameters of parathion and paraoxon uptake were determined in isolated and perfused rabbit and guinea pig lungs. They were related to organophosphate-induced lung cholinesterase inhibition. A single pass procedure was used to perfuse the lungs with an artificial medium perfusate containing paraoxon or parathion. The paraoxon and parathion concentrations were determined in the effluents collected at chosen intervals over an 18-min period beginning at the start of perfusion. Three inflowing concentrations (1 nmol/ml, 10 nmol/ml, and 20 nmol/ml) were tested in guinea pig lungs and one (10 nmol/ml) in rabbit lungs. Cholinesterase activity was determined at time 0 and at the end of the experiment. The lungs abundantly extracted paraoxon and parathion over the perfusion period. The extraction ratio was consistently greater in guinea pig than in rabbit lungs. The uptake velocity varied biexponentially in time, suggesting the existence of two compartments. Initial uptake velocities (A, B) and slopes (α and β) were calculated for both compartments. In guinea pigs, A, B and A + B increased proportionally to the supply rate of paraoxon and parathion while a and b remained constant. No significant difference was observed between parathion and paraoxon uptake kinetics. Parameter B was the only one to differ significantly between the two species (rabbits: 8.19 ± 1.53 for parathion and 6.85 ± 1.26 for paraoxon; guinea pigs: 12.75 ± 0.88 for parathion and 15.02 ± 3.84 for paraoxon). In the lungs of both species, there was a linear relation between y, the percentage of cholinesterase inhibition induced by either organophosphate, and X, the total amount of drug taken up by the lung tissue (in nmol/g/18 min). The following equations were obtained:y= 0.128x+ 0.979 (R2= 0.89,p< 0.001 for paraoxon);y= 0.120x− 6.57 (R2= 0.82,p< 0.005 for parathion). No difference was observed between the two organophosphates. After treatment with the cytochrome P450 inhibitor piperonyl butoxide, the above relations ceased to apply, but this treatment did not influence the kinetics of paraoxon and parathion uptake. The IC50 value calculated for paraoxon, i.e., the paraoxon concentration required to produce 50% inhibition of lung cholinesterase activity, was similar for guinea pigs (2.22 10−7± 0.22M) and rabbits (2.36 10−7± 0.24M). In conclusion, the biexponential evolution of the velocity of paraoxon and parathion uptake by the lungs thus demonstrates the presence of two pools. The lower extraction ratios calculated for rabbit lungs reflect the lower initial uptake velocity of the second compartment. In the range of concentrations investigated in guinea pigs, no saturable mechanism could be demonstrated for paraoxon and parathion. Cytochrome P450-related lung metabolic activity, through which parathion is converted to paraoxon, appears as a major step in parathion-induced lung cholinesterase inhibition, although it does not appear to affect parathion toxicokinetics.

Gender-differential liver plasma membrane affinities in hepatic tetrabromosulfonephthalein (TBS) uptake

The sex difference in the hepatic uptake of tetrabromosulfonephthalein (TBS) was investigated in male and female rats in two different experimental models. In the intact animal, the initial plasma disappearance constant rate, the initial velocity of uptake, and the plasma clearance of TBS were significantly higher in females than in males. In sinusoidal liver plasma membrane vesicles, kinetic parameters of TBS uptake were investigated in both sexes. The K m was lower in females than in males (5.5 ± 0.4 vs 17 ± 4 μM, P < 0.05), whereas V max showed comparable values (544 ± 15 vs 581 ± 60 nmol TBS/min/mg protein, mean ± SD, NS, females and males, respectively). Collectively, these data indicate that the sex difference in hepatic uptake of TBS is located at the sinusoidal liver plasma membrane and is due to a greater affinity of the electrogenic transport system(s) in females.

Hepatobiliary disposition of valproic acid and valproate glucuronide: Use of a pharmacokinetic model to examine the rate-limiting steps and potential sites of drug interactions

Previous work in this laboratory has suggested that the nonlinear disposition of valproic acid (VPA) in the rat may be due to nonlinear distribution of VPA into the liver. The present study was undertaken to elucidate further the hepatobiliary disposition of VPA. VPA (0.1-2 mmol/L) was incubated with isolated rat hepatocytes in vitro. Uptake of [(3)H]-VPA was linear from 10 to 50 seconds, with minimal (<7 percent) biotransformation. The initial velocity of VPA uptake varied in proportion with the extracellular concentration and was temperature independent, suggesting that VPA traverses the hepatocyte membrane predominantly by passive diffusion. In separate studies, the hepatobiliary disposition of VPA (20mg) was examined in the isolated perfused rat liver (IPL). A pharmacokinetic model was developed to describe the influence of phenobarbital on the hepatobiliary disposition of VPA and valproate glucuronide (V-G) in the IPL; all processes governing VPA and V-G disposition appeared to be linear. Acute administration of phenobarbital to the liver (1.12 mg) decreased the rate constant for canalicular egress of V-G (0.0489 +/- 0.0266 vs. 0.164 +/- 0.075 min(-1)). In vivo pretreatment with phenobarbital (75 mg/kg/d x 5 d) before liver isolation decreased the biliary excretion of both VPA (1.06E-04 +/- 0.27E-04 vs. 2.76E-04 +/- 0.45E-04 min(-1)) and V-G (5.63E- 03 +/- 1.98E-03 vs. 1.74E-02 +/- 0.5E-02 min(-1)), and increased the apparent volume of distribution of VPA (84.6 +/- 2.2 vs. 72.3 +/- 2.1 mL). In vivo phenobarbital pretreatment a changed V-G excretion from a formation to an elimination rate-limited process. These results are consistent with phenobarbital-associated impairment of canalicular egress of some organic anions. This work further supports the utility of pharmacokinetic modeling in: (1) determining the rate-limiting steps in hepatobiliary drug disposition and (2) identifying sites of drug interactions within the hepatobiliary system that may not be evident based on conventional mass-balance analysis.

Hepatobiliary disposition of valproic acid and valproate glucuronide: Use of a pharmacokinetic model to examine the rate-limiting steps and potential sites of drug interactions

Previous work in this laboratory has suggested that the nonlinear disposition of valproic acid (VPA) in the rat may be due to nonlinear distribution of VPA into the liver. The present study was undertaken to elucidate further the hepatobiliary disposition of VPA. VPA (0.1-2 mmol/L) was incubated with isolated rat hepatocytes in vitro. Uptake of [3H]-VPA was linear from 10 to 50 seconds, with minimal (<7 percent) biotransformation. The initial velocity of VPA uptake varied in proportion with the extracellular concentration and was temperature independent, suggesting that VPA traverses the hepatocyte membrane predominantly by passive diffusion. In separate studies, the hepatobiliary disposition of VPA (20mg) was examined in the isolated perfused rat liver (IPL). A pharmacokinetic model was developed to describe the influence of phenobarbital on the hepatobiliary disposition of VPA and valproate glucuronide (V-G) in the IPL; all processes governing VPA and V-G disposition appeared to be linear. Acute administration of phenobarbital to the liver (1.12 mg) decreased the rate constant for canalicular egress of V-G (0.0489 +/- 0.0266 vs. 0.164 +/- 0.075 min-1). In vivo pretreatment with phenobarbital (75 mg/kg/d x 5 d) before liver isolation decreased the biliary excretion of both VPA (1.06E-04 +/- 0.27E-04 vs. 2.76E-04 +/- 0.45E-04 min-1) and V-G (5.63E- 03 +/- 1.98E-03 vs. 1.74E-02 +/- 0.5E-02 min-1), and increased the apparent volume of distribution of VPA (84.6 +/- 2.2 vs. 72.3 +/- 2.1 mL). In vivo phenobarbital pretreatment a changed V-G excretion from a formation to an elimination rate-limited process. These results are consistent with phenobarbital-associated impairment of canalicular egress of some organic anions. This work further supports the utility of pharmacokinetic modeling in: (1) determining the rate-limiting steps in hepatobiliary drug disposition and (2) identifying sites of drug interactions within the hepatobiliary system that may not be evident based on conventional mass-balance analysis. (Hepatology 1996 Apr;23(4):771-80)

Confocal analysis of hepatocellular long-chain fatty acid uptake

Transmembrane transport and cytosolic accumulation of fatty acids were investigated using confocal laser scanning microscopy (cLSM). A Zeiss LSM 310 system was used to determine the uptake of the fluorescent fatty acid derivative 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3- diazol-4-yl)amino]octadecanoic acid (12-NBD stearate) (C18) in single rat hepatocytes. Uptake was a saturable process with a Michaelis-Menten constant value of 68 nM. Initial uptake velocity was dependent on extracellular presence of albumin and beta-lactoglobulin. Absence of albumin reduced uptake to 32 +/- 16% (P < 0.01) of control values. In the presence of unlabeled stearate, uptake of 12-NBD stearate was lowered to 49 +/- 12% (P < 0.01). Ion substitution experiments showed no sodium dependency of uptake. Increase in membrane potential led to a pronounced accumulation of the fatty acid derivative within the plasma membrane and in the adjacent cytoplasmic compartment, whereas membrane depolarization had no effect on uptake rates. In separate experiments line scans through representative hepatocytes were analyzed to generate "x-t" plots. 12-NBD stearate showed a fluorescence pattern with prominent staining of the area of the plasma membrane and the adjacent cytoplasm, dependent on the presence of extracellular albumin. For the hepatocellular cytosolic accumulation process of 12-NBD stearate a diffusion constant of 22.2 +/- 6.2 x 10(-9) cm2/s was calculated. In contrast to the long-chain fatty acid derivative 12-NBD stearate, short (C5)- and medium (C11)-chain fatty acids revealed no membrane interaction with hepatocytes. Erythrocytes also lacked a membrane interaction process for 12-NBD stearate. In conclusion, it was demonstrated that cLSM is capable of directly evaluating the cellular fatty acid uptake process at a subcellular level.

Relationship between the thyroid hormone transport system and the Na(+)-H+ exchanger in cultured rat brain astrocytes.

The entry of T3 and T4 into rat cultured astrocytes is mediated by a sterospecific saturable transport system. This study examines the effect of inhibiting the Na(+)-H+ exchanger and intracellular acidification on the initial velocity of [125I]T3 and [125I]T4 uptake. The resting intracellular pH (pHi) was approximately 7.15 in astrocytes exposed to CO2/HCO3(-)-free medium buffered with HEPES at pH 7.40 at 22 C. Isoosmotic replacement of extracellular sodium by mannitol or choline decreased the pHi by 0.15 pH unit and reduced uptake by about 20%. Replacing sodium with lithium had no effect on uptake. Amiloride, a specific blocker of the Na(+)-H+ exchanger, reduced pHi, as described above, and inhibited T3 and T4 uptake by about 35%. Acid loading the cells with a NH4+ pulse decreased the pHi by up to 1.2 pH units and the uptake of T3 and T4 by up to 50%. The maximum velocity of uptake was decreased, whereas the Km was unchanged. An isoosmotic increase in the extracellular K+ concentration to 50 mM had no effect on T3 uptake. The initial velocity of T3 uptake by acid-loaded cells was gradually restored by increasing the extracellular Na+ concentration. These results indicate that thyroid hormone transport into rat cultured astrocytes involves a mechanism linked to the activity of the Na(+)-H+ exchanger and the H+ concentration inside the cells.

Relationship between the thyroid hormone transport system and the Na(+)- H+ exchanger in cultured rat brain astrocytes

The entry of T3 and T4 into rat cultured astrocytes is mediated by a sterospecific saturable transport system. This study examines the effect of inhibiting the Na(+)-H+ exchanger and intracellular acidification on the initial velocity of [125I]T3 and [125I]T4 uptake. The resting intracellular pH (pHi) was approximately 7.15 in astrocytes exposed to CO2/HCO3(-)-free medium buffered with HEPES at pH 7.40 at 22 C. Isoosmotic replacement of extracellular sodium by mannitol or choline decreased the pHi by 0.15 pH unit and reduced uptake by about 20%. Replacing sodium with lithium had no effect on uptake. Amiloride, a specific blocker of the Na(+)-H+ exchanger, reduced pHi, as described above, and inhibited T3 and T4 uptake by about 35%. Acid loading the cells with a NH4+ pulse decreased the pHi by up to 1.2 pH units and the uptake of T3 and T4 by up to 50%. The maximum velocity of uptake was decreased, whereas the Km was unchanged. An isoosmotic increase in the extracellular K+ concentration to 50 mM had no effect on T3 uptake. The initial velocity of T3 uptake by acid-loaded cells was gradually restored by increasing the extracellular Na+ concentration. These results indicate that thyroid hormone transport into rat cultured astrocytes involves a mechanism linked to the activity of the Na(+)-H+ exchanger and the H+ concentration inside the cells.

Serotonin uptake and its kinetics in platelets of women with Alzheimer's Disease

The uptake of serotonin ( 14C-5-hydroxytryptamine, 5HT) in platelets and its kinetic characteristics were investigated in a group of women ( n=20) with probable Alzheimer's Disease (mean age = 76.0, years, SD = 8.27, range = 63–88) and in healthy normal women ( n = 18, mean age = 72.6 years, SD = 7.24, range = 61–84). Both the apparent affinity of binding of 5HT to the platelet membrane ( K m ) and the maximum velocity ( V max ) of the rate of transport of 14C-5HT into platelets were significantly higher in the Alzheimer's Disease group than in the normal comparison group. Initial velocity of 14C-5HT uptake nor passive diffusion (nonspecific uptake of 14C-5HT at 4 °C) differed significantly in the two groups. Our findings suggest abnormalities in the kinetic mechanisms involved in the uptake of 14C-5HT by platelets in women with Alzheimer's Disease.

Uptake of phenoxyacetic acid by Penicillium chrysogenum.

The uptake of phenoxyacetic acid by two different strains of Penicillium chrysogenum was studied. Phenoxyacetic acid (POA) was taken up by P. chrysogenum in a defined medium. Plots of initial velocity of POA uptake versus external substrate concentration, in the range 2-5000 microM, gave linear plots. Uptake of POA by induced and uninduced cells was identical. The initial velocity of POA uptake decreased as the pH of the suspension was increased from 5.4 to 7.2; the decrease closely paralleled the decline in the non-ionic form of the acid over this pH range. The initial velocity of POA uptake was not affected by the presence of phenylacetic acid. POA uptake proceeded until the cellular concentration was equal to the external concentration. It is concluded that POA is passively transported into P. chrysogenum by unmediated diffusion.

Evaluation of solitary pulmonary nodules with dynamic contrast-enhanced MR imaging--a promising technique.

The evaluation of a solitary pulmonary nodule (SPN) is one of the most frequently encountered challenges in thoracic radiology. In addition to a "state-of-the-art" evaluation of SPNs with CT and biopsy techniques, recently the assessment of the enhancement characteristics with iodinized contrast agents has shown its potential to improve the characterization of SPNs. We investigated whether dynamic contrast-enhanced MRI is suitable to assess the degree and kinetics of MR contrast enhancement and whether this technique could help in the noninvasive specification of SPNs. We studied prospectively 21 patients with SPNs. T1-weighted and proton density-weighted spoiled gradient-echo breath-hold images (2D-FLASH) were obtained before and after the administration of Gd-DTPA in a standard dosage of 0.1 mmol/kg body weight. The maximum enhancement and the initial velocity of contrast uptake were assessed and correlated with pathohistological findings. To quantify contrast enhancement, we used the relative signal intensity increase (S(rel)) and the recently introduced enhancement factor (EF) and contrast uptake equivalent (CE). Dynamic contrast-enhanced MRI proved to be well suited for the assessment of the contrast enhancement characteristics of SPNs. Significant differences were found in the degree and kinetics of contrast enhancement for specific types of nodules. Malignant neoplastic SPNs enhanced stronger and faster than benign neoplastic SPNs. The strongest and fastest enhancement, however, was found in a benign type of nodules where histology revealed inflammatory/fibrous lesions. These differences in contrast enhancement between the different pathohistological groups were more significant when EF and CE rather than S(rel) was used for the quantification of contrast enhancement. The results of this study indicate a potential role for dynamic contrast-enhanced MRI in the preoperative noninvasive evaluation of SPNs using EF and CE as contrast uptake assessment parameters.

Isolation and preliminary characterization of ferritin from clover seeds

Ferritin from clover (Trifolium subterraneum) seeds was isolated and characterized. It was shown to contain polypeptide units of 28–26.5 kDa. The apparent molecular mass of the native protein was 560 kDa. The average iron cores were 4 nm in diameter and contained 1300 iron atoms, with Fe:P = 4:1. Purified clover apoferritin was shown to be functional by means of iron uptake experiments. Plots of initial velocities of iron uptake (at pH 6.7) into clover and horse spleen apoferritins were found to have similar profiles.

NONLINEARITY OF HMG-COA REDUCTASE INHIBITOR, PRAVASTATIN IN BOTH HEPATIC UPTAKE AND BILIARY EXCRETION PROCESSES

Among the HMG-CoA reductase inhibitors, pravastatin which is hydrophilic in nature, has been shown to exhibit relatively specific inhibition of cholesterol synthesis in the liver. As one of the reasons for this relatively specific pharmacological activity, we demonstrated that the tissue distribution of pravastatin is limited because of its high hydrophilicity, while hepatic uptake by active transport mechanism via multispecific anion transporter takes place at the liver surface. We examined the hepatic elimination of HMG-CoA reductase inhibitor, pravastatin, at steady-state by measuring the plasma concentration in both arterial (Ca, ss) and hepatic venous (Chv, ss) blood. The hepatic extraction ratio at steady-state showed a clear decrease with increasing pravastatin infusion rate. The total hepatic elimination velocity (V) at steady-state exhibited the Michaelis-Menten type saturation with the mean logarithmic concentration (C), which is used in the parallel-tube model, and the values of Km and Vmax estimated from the plot of V vs. C were comparable with those obtained from the analysis of the initial uptake velocity using isolated hepatocytes. This result indicates that overall intrinsic clearance (CLint, all) was regulated by the uptake process, since pravastatin was efficiently taken up by an active transport mechanism followed by rapid metabolism and/or biliary excretion. Pravastatin is a typical drug whose clearance in the liver, which is the target organ in terms of its pharmacological effect as well as the major organ for its pharmacokinetics in the whole body, is governed mainly by an uptake process mediated by active transport.

Unbound ligand drives hepatocyte taurocholate and BSP uptake at physiological albumin concentration.

We have recently shown (D. Sorrentino, R.B. Robinson, C.-L. Kiang, and P.D. Berk. J. Clin. Invest. 84: 1325-1333, 1989) that, in a variety of isolated cell types, the uptake of oleate at physiological albumin concentrations is consistent with traditional pharmacokinetic theory (i.e., driven by unbound ligand). Lower albumin concentrations were associated with a deviant uptake pattern for which alternative theories have been proposed. Whether other classes of organic anions exhibit similar behavior is unknown. Therefore, we examined the effect of albumin on uptake of two widely studied organic anions, sulfobromophthalein (BSP) and taurocholate. Initial uptake velocity of [35S]BSP and [3H]taurocholate by isolated hepatocytes was studied employing a fixed albumin concentration and ligand-to-albumin molar ratios from 0.01:1 to 2:1 for taurocholate and 0.031:1 to 0.75:1 for BSP. In other experiments, albumin and ligand were altered in parallel, keeping their molar ratio constant. Unbound taurocholate concentrations were measured directly by equilibrium dialysis; unbound BSP concentrations were calculated from published data (K.J. Baker and S.E. Bradley. J. Clin. Invest. 45: 281-287, 1966). At 600 microM albumin, uptake of both ligands was a function of the unbound ligand concentration. At low ligand-to-albumin molar ratios and consequent unbound ligand concentrations this relationship was linear; over the entire range of unbound ligand concentrations studied, both ligands exhibited Michaelis-Menten kinetics, with definable maximal velocity and Michaelis constant values. At low albumin concentrations, the relationships between uptake and unbound ligand were unchanged for taurocholate; however, BSP exhibited altered kinetics similar to those observed with oleate. Nontraditional uptake kinetics at low albumin concentrations appear to correlate with very high affinity for albumin.

Effects of cocaine on sodium dependent dopamine uptake in rat striatal synaptosomes.

Initial velocity of uptake of dopamine (DA) has been measured in the presence of 1 microM cocaine as a function of both [DA] and [Na]. Although DA uptake is overwhelmingly dependent on sodium, it appears that a small amount of DA uptake takes place in the absence of sodium. This contrasts with a previous study of the sodium dependence of uptake without cocaine (referred to below as control), in which uptake was found to be 100% sodium dependent. The data were fitted to several rapid equilibrium models and the minimal best fit model identified. The interaction of transporter (C), DA (S), and Na+ (Na) in this present model is identical to the reaction scheme found previously to fit control data (no cocaine). Whereas the control model required translocation only as CNa2S, in the presence of cocaine (I), two additional translocated species are required to fit the data (CS and CNaS). Another previous study of the interaction of carrier and cocaine at a constant [Na]0 predicted that cocaine interacts with a transporter site other than the DA binding site and that uptake takes place as CS and CSI. The present results are consistent with the assumption that the CS and CNaS forms of the present model are actually CSI and CNaSI, since they are required to fit a model of the sodium dependence in the presence of cocaine, but are not required in the absence of cocaine.