Properties and regulation of organic cation transport in freshly isolated human proximal tubules.

The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.     

Characterization of 3-[(123)I]iodo-L-alpha-methyl tyrosine transport in astrocytes of neonatal rats

3-[(123)I]Iodo-L-alpha-methyl tyrosine ((123)I-IMT) is used for diagnosis and monitoring of brain tumours by means of single-photon emission tomography. As recently shown, (123)I-IMT is predominantly mediated into rat C6 glioma cells by sodium-independent system L for large neutral amino acids. Until now, (123)I-IMT transport in non-neoplastic glial cells has not been examined. Therefore, the aim of this study was to examine the cellular pathways and precise transport kinetics of (123)I-IMT uptake into astrocytes of neonatal rats. In particular sodium-independent (123)I-IMT transport into neonatal astrocytes was compared with sodium-independent (123)I-IMT uptake into neoplastic rat C6 glioma cells. Competitive inhibition experiments showed that (123)I-IMT is exclusively transported via sodium-independent system L into the neonatal astrocytes (92%). Kinetic analysis of sodium-independent (123)I-IMT uptake into neonatal astrocytes and into C6 glioma cells revealed apparent Michaelis constants K(M) = 13.9 +/- 0.5 microM and K(M) = 33.9 +/- 4.1 microM, respectively, which are in the same range of K(M) values as those recently determined for amino acid transport into neoplastic and non-neoplastic glial cells. Indeed, the K(M) values in the micromolar range correspond to the expression of the LAT-1 subunit of system L both in the neonatal astrocytes and in C6 glioma cells. However, sodium-independent maximum transport velocities (V(max)) differed significantly between neonatal astrocytes and C6 glioma cells (11.1 +/- 0.3 and 39.9 +/- 3.3 nmol/mg protein/10 min, respectively).     

Uptake of erythrocyte-associated component of blood testosterone and corticosterone to rat brain

To study transport of steroids by erythrocytes, the tissue uptake of erythrocyte-associated testosterone and corticosterone was studied in vivo using a single injection technique into the carotid artery of rats. A brain uptake index (BUI) was calculated by dividing the ratio of [3H]steroid to [14C]butanol (internal reference) in the brain tissue by that in the injection material, and multiplying by 100%. BUIs of testosterone and corticosterone in an erythrocyte suspension were 131 +/- 3% (mean +/- SE, n = 6) and 57.0 +/- 2.7% (n = 6), respectively, which were greater than those in buffer (100 +/- 4%; n = 4, P less than 0.01 and 39.8 +/- 4.6%; n = 4, P less than 0.01, respectively). The erythrocyte accounted for 83.9% and 76.7% of the total testosterone and corticosterone delivered to the tissues, respectively, when calculated on the assumption that the BUIs of steroid in buffer and in the supernatant of an erythrocyte suspension are the same. BUIs of corticosterone in hemolysate and in a suspension of erythrocyte plasma membranes (60.8 +/- 7.0%; n = 4 and 69.5 +/- 3.7%; n = 4, respectively) were also greater than those in buffer (P less than 0.05 and P less than 0.01, respectively). Our results suggest that the erythrocyte-associated component of testosterone and corticosterone are delivered to the tissue of rat brain, and that their membranes may play a major role in their capacity to transport steroids to the tissues.     

Effects of high myoplasmic L-lactate concentration on E-C coupling in mammalian skeletal muscle.

The effects of high myoplasmic L-lactate concentrations (20-40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca(2+) release, and passive Ca(2+) leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. L-Lactate (20 mM) significantly reduced maximum Ca(2+)-activated force by 4 +/- 0.5% (n = 5, P < 0.05) and 5 +/- 0.4% (n = 6, P < 0.05) for EDL and soleus, respectively. The Ca(2+) sensitivity was also significantly decreased by 0.06 +/- 0. 002 (n = 5, P < 0.05) and 0.13 +/- 0.01 (n = 6, P < 0.001) pCa units, respectively. Exposure to L-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 +/- 3% (n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 microM), or the chloride channel blocker anthracene-9-carboxylic acid (100 microM). L-Lactate (20 mM) increased passive Ca(2+) leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 +/- 5%, n = 9, P < 0.05) with no apparent effect in soleus fibers (100 +/- 2%, n = 3). These results indicate that the L-lactate ion per se has negligible effects on either voltage-dependent Ca(2+) release or SR Ca(2+) handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.     

Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells

The present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8,9- or 14,15-EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10(-7) M 14,15-EET: 120 +/- 7% of control; n = 6; P less than 0.025; 10(-6) M 14,15-EET: 145 +/- 10%; n = 20; P less than 0.0005; 10(-6) M 8,9-EET: 167 +/- 31%; n = 9; P less than 0.05), which was not affected by addition of the cyclooxygenase inhibitor indomethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14,15-EET administration induced intracellular alkalinization of 0.2-0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, greater than 85% of 3 mM 22Na uptake into mesangial cells was inhibited by 1 mM amiloride, indicating Na+/H+ exchange. Under these conditions, 14,15-EET stimulated Na+/H+ exchange by 42% and 8,9-EET stimulated Na+/H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo-inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14,15-EET. Twenty-four hours after addition of [14C]14,15-EET, greater than 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol. Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+/H+ exchange and mitogenesis in mesangial cells. These effects do not appear to be mediated via phospholipase C activation. In addition, 14,15-EET was selectively incorporated into cellular lipids known to mediate signal transduction. These observations extend the potential biologic roles of c-P450 arachidonate metabolites to include stimulation of cell proliferation and suggest a role for these compounds in vascular and renal injury.     

Evidence of saturable uptake mechanisms at maternal and fetal sides of the perfused human placenta by rapid paired-tracer dilution: studies with calcium and choline

Rapid uptake and efflux of 45Ca2+ and [3H]choline at the maternal and fetal interfaces of the syncytiotrophoblast in the dually-perfused human placenta was investigated by application of the single circulation paired-tracer dilution method (Yudilevich, Eaton, Short & Leichtweiss 1979). Cotyledons were perfused with Krebs-bicarbonate containing dextran (30 g/l; MW = 60-70,000) at 20 and 6 ml/min on maternal and fetal sides, respectively. The paired-tracer (test substrate and extracellular marker) technique consisted of an intra-arterial injection of a tracer bolus, followed by venous sampling over 5-6 min. There was a rapid (sec) uptake of 45Ca2+, followed by backflux (efflux into the ipsilateral circulation) which, over 5-6 min, was 59-100% on the fetal side. It was more variable but generally lower on the maternal interface. At 0.1 mM calcium, 45Ca2+ maximal uptake (Umax) was about 53% on the fetal side but on the maternal side it was variable and averaged 17%. At 2.4 mM calcium fetal side Umax was reduced to 40%. However, on the maternal side the effect was not consistent. Unidirectional influx (nmol/min per g) appeared to be not different on the two sides of the placenta. For [3H]choline (in choline-free perfusates) Umax was about 50% and 30% on fetal and maternal sides, respectively; tracer backflux was variable on the maternal side and averaged 50% on the fetal side. [3H]Choline uptake was highly inhibited by either 1.0 mM choline or the specific competitive inhibitor, hemicholinium-3 (0.1 mM). Specific transplacental transfer of 45Ca2+ (i.e. in excess of the extracellular marker) was not significant in either direction. For [3H]choline there was an apparent small excess (about 4%) preferential towards the fetal circulation. These findings in the human placenta are similar to those demonstrated previously in the guinea-pig placenta which suggested the existence of specific transport systems for choline and calcium on both sides of the syncytiotrophoblast.     

Cell membrane transport of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the liver and systemic bioavailability.

Modulation of hepatic disposition of MPTP could influence susceptibility to its neurotoxicity. Therefore, we studied hepatocellular transport of MPTP in the perfused rat liver and isolated rat hepatocytes. The perfused liver extensively extracted MPTP. Amiloride and tubocurarine, inhibitors of OCT1, increased MPTP recovery (253 +/- 78 and 283 +/- 64%, respectively) and reduced PS(influx) (0.69 +/- 0.36 to 0.27 +/- 0.11, and 0.97 +/- 0.50 to 0.23 +/- 0.05 ml/s/g, respectively). P-glycoprotein inhibitor, daunomycin, and Oatp 1 & 2 inhibitor, rifamycin, had no effect. In isolated hepatocytes, amiloride and tubocurarine increased hepatic uptake of MPTP (23 +/- 12 and 6 +/- 2%, respectively). Daunomycin reduced MPTP uptake by 22 +/- 8% and rifamycin had no effect. Only a small proportion of MPTP is taken up into hepatocytes by transporters; however, modulation of these transport mechanisms will influence systemic bioavailability.     

Age-dependent effects induced by ouabain in rat heart muscles and cellular Ca2+ concentration

Responsiveness to ouabain of the inotropic and chronotropic effects in rat atrial muscles during development (3-18 wks old) was examined. In spontaneously beating rat right atrial muscles, ouabain (3-30 microM) caused a potent positive inotropic effect in a concentration-dependent manner, but failed to have a chronotropic effect; at 30 microM, 78.6 +/- 3.4% (n = 14, p<0.01) in the contractile force and -1.1 +/- 2.3% (n = 14, p>0.05) in the sinus rate in 10-wk-old rats. The myocardium during development increased the responsiveness to ouabain (10 microM) by 27.6 +/- 2.1% (n = 14, p<0.01), 58.7 +/- 3.3% (n = 14, p<0.001), and 47.2 +/- 2.3% (n = 14, p<0.001) in 3-, 10-, and 18- wk-old rats, respectively. However, the response on the sinus rate was not modified in all of the developing stages. Higher frequencies of stimulation caused the more potent inotropic effect in left atrial muscles. In the experiments using a Ca2+-sensitive fluorescent dye (Fura-2), ouabain (10 and 30 microM) increased the cellular Ca2+ concentrations by 3.0 +/- 2.1% (n = 6, p>0.05) and 12.7 +/- 1.5% (n = 6, p<0.05) in 3-wk-old rats and by 13.0 +/- 2.7% (n = 6, p<0.05) and 42.9 +/- 3.1% (n = 6, p<0.01) in 18-wk-old rats, respectively. These results suggest that the ouabain-evoked response is enhanced during development (but tends to decrease from the maximum after maturing), presumably resulting from developmental degrees of cellular mechanisms such as Na+/K+ pump activity and Na+/Ca2+ exchange and is reflected by changes in the cellular Ca2+ concentration.     

Defective suppressor cell function mediated by T8+ cell lines from patients with progressive multiple sclerosis

Activated suppressor cell function, induced with either concanavalin A or OKT3 and mediated by either unfractionated mononuclear cells or "panning" enriched T8+ cells, freshly isolated from peripheral blood, is reduced in patients with progressive multiple sclerosis (MS) as compared with control donors. In this study, we generated T8+ cell lines from the peripheral blood of these same patients and controls. Suppressor activity, mediated by T8+ cells exposed to OKT3 on days 1, 7, and 14 of culture and then treated with mitomycin C on day 16, was significantly reduced in the MS group (mean percent suppression 13% +/- 5) as compared with the control group (68% +/- 6, n = 8, p less than 0.001). No differences were noted in [3H]thymidine uptake by the OKT3-stimulated T8+ cell lines of MS and control groups. Mean percent suppression mediated by T4+ cell lines did not differ between MS and control groups (15% +/- 4, n = 3, vs 22% +/- 2, n = 4). These current data suggest that the previously observed defect in T8+ cell-mediated activated suppressor cell function in MS is a persistent one, favoring the postulate that the defect reflects intrinsic alterations in this cell population rather than a transient effect of serum factors on T8+ cell function.     

Evidence that activation of 2-deoxy-D-glucose transport in rat thymocyte suspensions results from enhanced coupling between transport and hexokinase activity.

1. Suspensions of rat thymocytes accumulate free 2-deoxy-D-glucose (2-dGlc) within the cytosol to a concentration approx. 25-fold above the external concentration. This active accumulation was enhanced by 40 nM-phorbol 12-myristate 13-acetate (phorbol). 2. The Km for zero-trans uptake in control cells was 2.3 +/- 0.14 mM and Vmax. was 0.41 +/- 0.08 mumol/min per 10(10) cells (n = 6). In cells treated with phorbol (40 nM) the Km for zero-trans uptake was 1.2 +/- 0.13 mM and Vmax. 0.46 +/- 0.03 mumol/min per 10(10) cells (n = 6). The Km was decreased significantly by phorbol (P less than 0.01). 3. Phorbol-dependent activation of thymocytes delayed exit of free 2-dGlc into sugar-free solution and prevented exchange exit. Activation had no effect on 3-O-methyl D-glucoside (3-OMG) exit. 4. Coupling of 2-dGlc transport to hexokinase activity was determined by observing the effects of various concentrations of unlabelled cytosolic 2-dGlc on influx of labelled 2-dGlc into the hexose phosphate pool. In control cells this coupling was 0.81 +/- 0.02 and in phorbol-activated cells it was 0.92 +/- 0.01 (P less than 0.01). 5. The high-affinity inhibitor of hexokinase, mannoheptulose, inhibited uptake of 2-dGlc in both control and phorbol-treated cells. These data are consistent with a model for activation of sugar transport in which hexokinase activity is integrated with the sugar transporter at the endofacial surface. The results suggest that phorbol increases the degree of coupling transport with hexokinase activity, thereby leading to an increase in the rate of uptake of 2-dGlc, a decrease in exit of free 2-dGlc from the cytosol and an increase in free 2-dGlc accumulation.     

Effect of insulin and glucagon on the uptake of carnitine by perfused rat liver

The possible direct effects of insulin and glucagon on carnitine uptake by perfused rat liver were studied with L-[3H]carnitine of an initial concentration of 50 microM in the perfusate. Insulin (10 nM) did not significantly affect the uptake by livers from fed animals. However, insulin could reverse the stimulated transport by livers from 24-h fasted animals, reducing the uptake rate from 852 +/- 54.1 to 480 +/- 39.9 (mean +/- S.E.), P less than 0.01 (rates are expressed as nmol per h per 100 g body wt). Glucagon (50 nM) stimulated the uptake rate when livers were either from fed (551 +/- 40.1 vs. 915 +/- 55.3, P less than 0.01) or from fasted animals (852 +/- 54.1 vs. 1142 +/- 88.1, P less than 0.02). Based on these and earlier observations, we propose that the carnitine concentration in rat liver is controlled by insulin and glucagon via cellular transport processes.     

Nucleoside transport in rat cerebral-cortical synaptosomes. Evidence for two types of nucleoside transporters.

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.     

Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit.

In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.     

Neural regulation of intestinal smooth muscle growth in vitro

The loss of intrinsic neurons is an early event in inflammation of the rat intestine that precedes the growth of intestinal smooth muscle cells (ISMC). To study this relationship, we cocultured ISMC and myenteric plexus neurons from the rat small intestine and examined the effect of scorpion venom, a selective neurotoxin, on ISMC growth. By 5 days after neuronal ablation, ISMC number increased to 141+/-13% (n = 6) and the uptake of [(3)H]thymidine in response to mitogenic stimulation was nearly doubled. Atropine caused a dose-dependent increase in [(3)H]thymidine uptake in cocultures, suggesting the involvement of neural stimulation of cholinergic receptors in regulation of ISMC growth. In contrast, coculture of ISMC with sympathetic neurons increased [(3)H]thymidine uptake by 45-80%, which was sensitive to propranolol (30 microM) and was lost when the neurons were separated from ISMC by a permeable filter. Western blotting showed that coculture with myenteric neurons increased alpha-smooth muscle-specific actin nearly threefold to a level close to ISMC in vivo. Therefore, factors derived from enteric neurons maintain the phenotype of ISMC through suppression of the growth response, whereas catecholamines released by neurons extrinsic to the intestine may stimulate their growth. Thus inflammation-induced damage to intestinal innervation may initiate or modulate ISMC hyperplasia.     

Tempol reduces infarct size in rodent models of regional myocardial ischemia and reperfusion.

Reactive oxygen species (ROS) contribute to ischemia-reperfusion injury of the heart. This study investigates the effects of tempol, a membrane-permeable radical scavenger on (i) the infarct size caused by regional myocardial ischemia and reperfusion of the heart in vivo (rat, rabbit) and in vitro (rat), and (ii) the cell injury caused by hydrogen peroxide (H2O2) in rat cardiac myoblasts (H9c2 cells). In the anesthetized rat, tempol reduced the infarct size caused by regional myocardial ischemia (25 min) and reperfusion (2 h) from 60 +/- 3% (control, n = 8) to 24 +/- 5% (n = 6, p < .05). In the anesthetized rabbit, tempol also attenuated the infarct size caused by myocardial ischemia (45 min) and reperfusion (2 h) from 59 +/- 3% (control, n = 6) to 39 +/- 5% (n = 5, p < .05). Regional ischemia (35 min) and reperfusion (2 h) of the isolated, buffer-perfused heart of the rat resulted in an infarct size of 54 +/- 4% (control n = 7). Reperfusion of hearts with buffer containing tempol (n = 6) caused a 37% reduction in infarct size (n = 6, p < .05). Pretreatment of rat cardiac myoblasts with tempol attenuated the impairment in mitochondrial respiration caused by H2O2 (1 mM for 4 h). Thus, the membrane-permeable radical scavenger tempol reduces myocardial infarct size in rodents.     

Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.     

Lactate transport activity in rat skeletal muscle sarcolemmal vesicles after acute exhaustive exercise.

The effect of a single bout of exhaustive exercise on muscle lactate transport capacity was studied in rat skeletal muscle sarcolemmal (SL) vesicles. Rats were assigned to a control (C) group (n = 14) or an acutely exercised (E) group (n = 20). Exercise consisted of treadmill running (25 m/min, 10% grade) to exhaustion. SL vesicles purified from C and E rats were sealed because of sensitivity to osmotic forces. The time course of 1 mM lactate uptake in zero-trans conditions showed that the equilibrium level in the E group was significantly lower than in the C group (P < 0.05). The initial rate of 1 mM lactate uptake decreased significantly from 2.44 +/- 0.22 to 1.03 +/- 0.08 nmol. min(-1). mg protein(-1) (P < 0.05) after exercise, whereas that of 50 mM lactate uptake did not differ significantly between the two groups. For 100 mM external lactate concentration ([lactate]), exhaustive exercise increased initial rates of lactate uptake (219.6 +/- 36.3 to 465.4 +/- 80.2 nmol. min(-1). mg protein(-1), P < 0.05). Although saturation kinetics were observed in the C group with a maximal transport velocity of 233 nmol. min(-1). mg protein(-1) and a Michealis-Menten constant of 24.5 mM, saturation properties were not seen after exhaustive exercise in the E group, because initial rates of lactate uptake increased linearly with external [lactate]. We conclude that a single bout of exhaustive exercise significantly modified SL lactate transport activity, resulting in a decrease in 1 mM lactate uptake and was associated with alterations in the saturable properties at [lactate] above 50 mM. These results suggest that changes in sarcolemmal lactate transport activity may alter lactate and proton exchanges after exhaustive exercise.     

Decreased responsiveness of gluconeogenesis to the modulation by sulfonylureas in hepatocytes isolated from obese (fa/fa) Zucker rats

The influence of the hypoglycemic agent glipizide (0-100 microM) on the rate of gluconeogenesis from lactate, as well as on the levels of fructose 2,6-bisphosphate, has been investigated in hepatocytes isolated from genetically obese (fa/fa) Zucker rats and from their corresponding lean (Fa/-) littermates. As compared to lean rat hepatocytes, liver cells isolated from obese animals showed a lower rate of basal gluconeogenesis (0.9 +/- 0.2 vs 5.4 +/- 0.5 micromol of lactate converted to glucose/g cell x 30 min, n=4) and higher levels of fructose 2,6-bisphosphate (11.5 +/- 1.0 vs 5.9 +/- 0.4 nmol/g cell, n=8-9). In lean rat hepatocytes, the presence of glipizide in the incubation medium caused a dose-dependent inhibition of the rate of lactate conversion to glucose (maximal inhibition=46%; EC50 value=26 microM), and simultaneously raised the cellular content of fructose-2,6-bisphosphate (maximal increment=40%; EC50 value=10 microM). In contrast, in hepatocytes isolated from obese rats, the inhibition of gluconeogenesis and the increment in fructose-2,6-bisphosphate levels elicited by glipizide were significantly reduced (maximal effects of 22 and 13%, respectively). Similarly, the activation of glycogen phosphorylase and the increase in hexose 6-phosphate levels in response to glipizide were less marked in obese rat hepatocytes than in liver cells isolated from lean animals. These results demonstrate that the efficacy of sulfonylureas as inhibitors of hepatic gluconeogenesis is reduced in the genetically obese (fa/fa) Zucker rat.     

The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.     

Evidence for a close link between the thyroid hormone transport system and the aromatic amino acid transport system T in erythrocytes

The transport of [125I]triiodothyronine ([125I]T3) and [3H]tryptophan ([3H]Trp) by washed rat erythrocytes was studied at 25 degrees C in the presence of leucine in order to block the neutral amino acid transport system L. Eadie-Hofstee plots of initial velocity data gave the following values of Km (micromolar) and Vmax (nanomole/min/10(8) cells): 0.122 +/- 0.007 and 0.140 +/- 0.021 for T3, and 558 +/- 28 and 17.4 +/- 2.3 for Trp (n = 5). The Trp transport system in rat erythrocytes is similar to the human erythrocyte aromatic amino acid-specific system T described by Rosenberg et al. (Rosenberg, R., Young, J. D., and Ellory, J. C. (1980) Biochim. Biophys. Acta 598, 375-384). Unlabeled aromatic amino acids (e.g. Trp, phenylalanine, tyrosine) competitively inhibited [125I]T3 uptake and unlabeled iodothyronine analogues (e.g. T3, D-T3, thyroxine, thyronine) competitively inhibited [3H]Trp uptake. The inhibition constants of these competitors measured with each labeled substrate were highly correlated. N-Ethylmaleimide irreversibly inhibited T3 and Trp transport and each substrate protected the transport system of the other from inactivation by N-ethylmaleimide. The Vmax of T3 and Trp transport by human erythrocytes were 500 and 120 times lower, respectively, than those of rat erythrocytes (0.30 and 126 pmol/min/10(8) cells, respectively). The T3 and Trp transport activities of sheep erythrocytes were undetectable. These results indicate that T3 and Trp either share a common multi-specific transport system or are transported by closely linked systems which interact in the erythrocyte membrane.