Creatine transporter activity and content in the rat heart supplemented by and depleted of creatine

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CrT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog beta-guanidinopropionic acid (beta-GPA). Creatine transport in control hearts followed saturation kinetics with a K(m) of 70 +/- 13 mM and a V(max) of 3.7 +/- 0.07 nmol x min(-1) x g wet wt(-1). Creatine supplementation significantly decreased the V(max) of the CrT (2.7 +/- 0.17 nmol x min(-1) x g wet wt(-1)). This was matched by an approximately 35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed beta-GPA exhibited a >80% decrease in tissue creatine and increase in beta-GPA(total). The V(max) of the CrT was increased (6.0 +/- 0.25 nmol x min(-1) x g wet wt(-1)) and the K(m) decreased (39.8 +/- 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool.     

Study on the effect of hypergravity stress on L-glutamate uptake by rat brain nerve endings

Using rat brain synaptosomes, we have investigated the effect of hypergravity on the kinetic parameters of Na(+)-dependent, high-affinity L-glutamate transport activity. The time-course of L-[14C]-glutamate uptake and dependence of L-[14C]-glutamate uptake velocity on glutamate concentrations were analyzed. K(m) and Vmax of this process have been determined. The hypergravity stress was created by centrifugation of rats for 1 hour at 10 g. We observed no differences in K(m) values between the control rats (10.7 +/- 2.5 microM) and animals exposed to hypergravity (6.7 +/- 1.5 microM). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate was not sensitive to hypergravity stress. In contrast, the maximal velocity of glutamate uptake changed in hypergravity conditions. Vmax reduced from 12.5 +/- +/- 3.2 nmol/min per 1 mg of protein (control group) to 5.6 +/- 0.9 nmol/min per 1 mg of protein (animals, exposed to hypergravity stress). The possible mechanisms of attenuation of the glutamate transporter activity without modifying K(m) of glutamate uptake were discussed.     

Cyclosporin A inhibits creatine uptake by altering surface expression of the creatine transporter

The immunosuppressive drug cyclosporin A (CsA) inhibited the hCRT-1 cDNA-induced creatine uptake in Xenopus oocytes and the endogenous creatine uptake in cultured C(2)C(12) muscle cells in a dose- and time-dependent manner. FK506, another potent immunosuppressant, was unable to mimic the effect of CsA suggesting that the inhibitory effect of CsA was specific. To delineate the mechanism underlying, we investigated the effect of CsA on the K(m) and V(max) of creatine transport and also on the cell surface distribution of the creatine transporter. Although CsA treatment did not affect the K(m) (20-24 microm) for creatine, it significantly decreased the V(max) of creatine uptake in both oocytes and muscle cells. CsA treatment reduced the cell surface expression level of the creatine transporter in the muscle cells by approximately 60% without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in creatine uptake. Taken together, our results suggest that CsA inhibited creatine uptake by altering the surface abundance of the creatine transporter. We propose that CsA impairs the targeting of the creatine transporter by inhibiting the function of an associated cyclophilin, resulting in an apparent loss in surface expression of the creatine transporter. Our results also suggest that prolonged exposure to CsA may result in chronically creatine-depleted muscle, which may be a cause for the development of CsA-associated clinical myopathies in organ transplant patients.     

Absence of acute doxorubicin-induced dysfunction of heart mitochondrial oxidative phosphorylation and creatine kinase activities

Since reductions in cardiac high-energy phosphate content and dysfunction of mitochondrial activities have been demonstrated after doxorubicin exposure, one mechanism of doxorubicin cardiotoxicity has been thought to be an interference with mitochondrial energy metabolism. To determine whether mitochondrial dysfunction is induced by acute drug exposure, isolated rat hearts were perfused with 10(-5) M doxorubicin for 70 min followed by mitochondrial isolation. Rates of electron transport, creatine kinase activity, acceptor control, respiratory control, and ADP/O ratios were assayed and correlated to doxorubicin-induced abnormalities in left ventricular function. At doses of doxorubicin sufficient to cause a marked deterioration of left ventricular systolic pressure and a rise in end-diastolic pressure, no decreases were noted in the measured mitochondrial parameters with either glutamate plus malate or succinate as respiratory substrates. In fact, in some cases the rates of electron transport were higher in mitochondria isolated from the treated hearts. In addition, isolated heart mitochondria were directly incubated in doxorubicin at doses as high as 10(-4) M for up to 70 min at 0 and 20 degrees C and 1.5 min at 37 degrees C. Under these conditions functional impairment of mitochondrial respiration was also not detected. Therefore, it appears that acute doxorubicin cardiotoxicity cannot be related to primary mitochondrial defects in high-energy phosphate metabolism. These data lend further support to the notion that doxorubicin cardiotoxicity may be fundamentally related to changes in coronary vascular resistance and resultant damage induced by hypoperfusion.     

Creatine Transport in Cultured Cells of Rat and Mouse Brain

Astroglia-rich cultures derived from brains of newborn rats or mice use a transport system for the uptake of creatine. The uptake system is saturable, Na+-dependent, and highly specific for creatine and Na+. Kinetic studies on rat cells revealed a Km value for creatine of 45 microM, a Vmax of 17 nmol x h-1 x (mg of protein)-1, and a Km value of 55 mM for Na+. The carrier is competitively inhibited by guanidinopropionate (Ki = 15 microM). No such transport system was found in neuron-rich primary cultures from embryonic rat brain. It is hypothesized that creatine transport is an astroglial rather than a neuronal function.     

Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin.

The quinonoid anthracycline, doxorubicin (Adriamycin) is a potent anti-neoplastic agent whose clinical use is limited by severe cardiotoxicity. Mitochondrial damage is a major component of this cardiotoxicity, and rival oxidative and non-oxidative mechanisms for inactivation of the electron transport chain have been proposed. Using bovine heart submitochondrial preparations (SMP) we have now found that both oxidative and non-oxidative mechanisms occur in vitro, depending solely on the concentration of doxorubicin employed. Redox cycling of doxorubicin by Complex I of the respiratory chain (which generates doxorubicin semiquinone radicals, O2-, H2O2, and .OH) caused a 70% decrease in the Vmax. for NADH dehydrogenase during 15 min incubation of SMP, and an 80% decrease in NADH oxidase activity after 2 h incubation. This inactivation required only 25-50 microM-doxorubicin and represents true oxidative damage, since both NADH (for doxorubicin redox cycling) and oxygen were obligatory participants. The damage appears localized between the NADH dehydrogenase flavin (site of doxorubicin reduction) and iron-sulphur centre N-1. Succinate dehydrogenase, succinate oxidase, and cytochrome c oxidase activities were strongly inhibited by higher doxorubicin concentrations, but this phenomenon did not involve doxorubicin redox cycling (no NADH or oxygen requirement). Doxorubicin concentrations of 0.5 mM were required for 50% decreases in these activities, except for cytochrome c oxidase which was only 30% inhibited following incubation with even 1.0 mM-doxorubicin. Our results indicate that low concentrations of doxorubicin (50 microM or less) can catalyse a site-specific oxidative damage to the NADH oxidation pathway. In contrast, ten-fold higher doxorubicin concentrations (or more) are required for non-oxidative inactivation of the electron transport chain; probably via binding to cardiolipin and/or generalized membrane chaotropic effects. The development of agents to block doxorubicin toxicity in vivo will clearly require detailed clinical studies of doxorubicin uptake in the heart.     

Regulation of pantothenic acid transport in the heart. Involvement of a Na+-cotransport system

Pantothenic acid transport was studied in the isolated perfused rat heart and isolated sheep cardiac sarcolemmal vesicles. In the perfused heart, pantothenic acid transport was significantly greater if hearts were perfused as working hearts rather than Langendorff hearts, but was unaffected by the perfusion substrates used (11 mM glucose or 1.2 mM palmitate). Uptake rates of pantothenic acid in working hearts are dependent on perfusate concentrations of pantothenic acid (a Vmax of 418 nmol/g dry weight/30 min and a Km for pantothenic acid of 10.7 mircoM were obtained). Reduction in perfusate Na+ concentration from 145 to 105 mM (the Na+ was replaced with 40 mM choline) resulted in a small but significant decrease in pantothenic acid uptake. At 145 mM Na+, addition of a mixture of amino acids, whose uptake is Na+-dependent, resulted in a significant decrease in pantothenic acid uptake by the heart (173 +/- 5 to 132 +/- 12 nmol/g dry weight). If an inward Na+ gradient in isolated, purified sarcolemmal vesicles, was imposed, a rapid uptake of pantothenic acid was observed. Uptake rates are markedly reduced if Na+ was replaced by equimolar concentrations of K+ or if external Na+ was reduced below 40 mM. In the presence of Na+, increasing pantothenic acid concentrations resulted in an increase in pantothenic acid uptake by the vesicles. Combined, these data demonstrate that pantothenic acid is transported across the myocardial sarcolemmal membrane by a Na+-dependent mechanism, which may be common to a number of small molecules.     

Diaphragm and cardiac mitochondrial creatine kinases are impaired in sepsis.

Previous studies indicate that ATP formation by the electron transport chain is impaired in sepsis. However, it is not known whether sepsis affects the mitochondrial ATP transport system. We hypothesized that sepsis inactivates the mitochondrial creatine kinase (MtCK)-high energy phosphate transport system. To examine this issue, we assessed the effects of endotoxin administration on mitochondrial membrane-bound creatine kinase, an important trans-mitochondrial ATP transport system. Diaphragms and hearts were isolated from control (n = 12) and endotoxin-treated (8 mg.kg(-1).day(-1); n = 13) rats after pentobarbital anesthesia. We isolated mitochondria using techniques that allow evaluation of the functional coupling of mitochondrial creatine kinase MtCK activity to oxidative phosphorylation. MtCK functional activity was established by 1) determining ATP/creatine-stimulated oxygen consumption and 2) assessing total creatine kinase activity in mitochondria using an enzyme-linked assay. We examined MtCK protein content using Western blots. Endotoxin markedly reduced diaphragm and cardiac MtCK activity, as determined both by ATP/creatine-stimulated oxygen consumption and by the enzyme-linked assay (e.g., ATP/creatine-stimulated mitochondrial respiration was 173.8 +/- 7.3, 60.5 +/- 9.3, 210.7 +/- 18.9, was 67.9 +/- 7.3 natoms O.min(-1).mg(-1) in diaphragm control, diaphragm septic, cardiac control, and cardiac septic samples, respectively; P < 0.001 for each tissue comparison). Endotoxin also reduced diaphragm and cardiac MtCK protein levels (e.g., protein levels declined by 39.5% in diaphragm mitochondria and by 44.2% in cardiac mitochondria; P < 0.001 and P = 0.009, respectively, comparing sepsis to control conditions). Our data indicate that endotoxin markedly impairs the MtCK-ATP transporter system; this phenomenon may have significant effects on diaphragm and cardiac function.     

Glucose uptake in vivo in skeletal muscles of insulin-injected chicks

Glucose uptake across the plasma membrane in animal cells plays a crucial role in whole-body glucose homeostasis. Insulin-stimulated glucose transport activity in vivo in several tissues was estimated using the 2-deoxy-D-[1-(3)H]glucose ([(3)H]2DG) uptake determination method. A tracer dose of [(3)H]2DG was injected intravenously into 8-day-old chicks (Gallus gallus) administered simultaneously or previously with porcine insulin (40 microg/kg BW). After 10 or 20 min, several major tissues, including skeletal and cardiac muscle, were sampled and their 2-deoxy-D-[1-(3)H]glucose 6-phosphate content analyzed. Plasma glucose concentration and [(3)H]2DG radioactivity were lowered by insulin within 20 min of [(3)H]2DG administration, while the plasma [(3)H]2DG/glucose ratio was not significantly different between chicks injected with insulin and their control counterparts. A marked uptake of 2DG was observed in cardiac tissue and brain, followed by kidney and skeletal muscles. In skeletal muscles, insulin increased the 2DG uptake in soleus, extensor digitorum longus and pectoralis superficialis muscles. On the other hand, no significant increases in insulin-induced 2DG uptake were detected in cardiac muscle or adipose tissue compared to controls. The results show that glucose transport across the plasma membrane in vivo in most skeletal muscles tested, but not cardiac muscle, was increased by insulin administration to chicks. These findings suggest that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack GLUT4 homologous gene, the insulin-responsive glucose transporter in mammals.     

AMPK and substrate availability regulate creatine transport in cultured cardiomyocytes

Profound alterations in myocellular creatine and phosphocreatine levels are observed during human heart failure. To maintain its intracellular creatine stores, cardiomyocytes depend upon a cell membrane creatine transporter whose regulation is not clearly understood. Creatine transport capacity in the intact heart is modulated by substrate availability, and it is reduced in the failing myocardium, likely adding to the energy imbalance that characterizes heart failure. AMPK, a key regulator of cellular energy homeostasis, acts by switching off energy-consuming pathways in favor of processes that generate energy. Our objective was to determine the effects of substrate availability and AMPK activation on creatine transport in cardiomyocytes. We studied creatine transport in rat neonatal cardiomyocytes and HL-1 cardiac cells expressing the human creatine transporter cultured in the presence of varying creatine concentrations and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Transport was enhanced in cardiomyocytes following incubation in creatine-depleted medium or AICAR. The changes in transport were due to alterations in V(max) that correlated with changes in total and cell surface creatine transporter protein content. Our results suggest a positive role for AMPK in creatine transport modulation for cardiomyocytes in culture.     

Nomega-nitro-L-arginine methylester ameliorates myocardial toxicity induced by doxorubicin

The effects of Nomega-nitro-L-arginine methylester (L-NAME) and L-arginine on cardiotoxicity that is induced by doxorubicin (Dox) were investigated. A single dose of Dox 15 mg/kg i.p. induced cardiotoxicity, manifested biochemically by a significant elevation of serum creatine phosphokinase (CPK) activity [EC 2.7.3.2]. Moreover, cardiotoxicity was further confirmed by a significant increase in lipid peroxides, measured as malon-di-aldehyde (MDA) in cardiac tissue homogenates. The administration of L-NAME 4 mg/kg/d p.o. in drinking water 5 days before and 3 days after the Dox injection significantly ameliorated the cardiotoxic effects of Dox, judged by the improvement in both serum CPK activity and lipid peroxides in the cardiac tissue homogenates. On the other hand, the administration of L-arginine 70 mg/kg/d p.o. did not protect the cardiac tissues against the toxicity that was induced by the Dox treatment. The findings of this study suggest that L-NAME can attenuate the cardiac dysfunction that is produced by the Dox treatment via the mechanism(s), which may involve the inhibition of the nitric oxide (NO) formation. L-NAME may, therefore, be a beneficial remedy for cardiotoxicity that is induced by Dox and can then be used to improve the therapeutic index of Dox.     

The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease

The organic cation transporter (OCTN1) plays key roles in transport of selected organic cations, but understanding of its biological functions remains limited by restricted knowledge of its substrate targets. Here we show capacity of human OCTN1-reconstituted proteoliposomes to mediate uptake and efflux of [(3)H]acetylcholine, the Km of transport being 1.0mM with V(max) of 160nmol?mg(-1)protein?min(-1). OCTN1-mediated transport of this neurotransmitter was time-dependent and was stimulated by intraliposomal ATP. The transporter operates as uniporter but translocates acetylcholine in both directions. [(3)H]acetylcholine uptake was competitively inhibited by tetraethylammonium, γ-butyrobetaine and acetylcarnitine, and was also inhibited by various polyamines. Decreasing intraliposomal ATP concentrations increased OCTN Km for acetylcholine, but V(max) was unaffected. Evaluation of the acetylcholine transporter properties of a variant form of OCTN1, the Crohn's disease-associated 503F variant, revealed time course, Km and V(max) for acetylcholine uptake to be comparable to that of wild-type OCTN1. Km for acetylcholine efflux was also comparable for both OCTN1 species, but V(max) of OCTN1 503F-mediated acetylcholine efflux (1.9nmol?mg(-1)protein?min(-1)) was significantly lower than that of wild-type OCTN1 (14nmol?mg(-1)protein?min(-1)). These data identify a new transport role for OCTN1 and raise the possibility that its involvement in the non-neuronal acetylcholine system may be relevant to the pathogenesis of Crohn's disease.     

Creatine uptake and creatine transporter expression among rat skeletal muscle fiber types

Total creatine (Cr(total) = phosphocreatine + creatine) concentrations differ substantially among mammalian skeletal muscle. Because the primary means to add Cr(total) to muscle is uptake of creatine through the sodium-dependent creatine transporter (CrT), differences in creatine uptake and CrT expression could account for the variations in [Cr(total)] among muscle fiber types. To test this hypothesis, hindlimbs of adult rats were perfused with 0.05-1 mM [(14)C]creatine for up to 90 min. Creatine uptake rates at 1 mM creatine were greatest in the soleus (140 +/- 8.8 nmol x h(-1) x g(-1)), less in the red gastrocnemius (117 +/- 8.3), and least in the white gastrocnemius (97 +/- 10.7). These rates were unaltered by time, insulin concentration, or increased perfusate sodium concentration. Conversely, creatine uptake rates were correspondingly decreased among fiber types by lower creatine and sodium concentrations. The CrT protein content by Western blot analysis was similarly greatest in the soleus, less in the red gastrocnemius, and least in the white gastrocnemius, whereas CrT mRNA was not different. Creatine uptake rates differ among skeletal muscle fiber sections in a manner reasonably assigned to the 58-kDa band of the CrT. Furthermore, creatine uptake rates scale inversely with creatine content, with the lowest uptake rate in the fiber type with the highest Cr(total) and vice versa. This suggests that the creatine pool fractional turnover rate is not common across muscle phenotypes and, therefore, is differentially regulated.     

Resveratrol treatment protects against doxorubicin-induced cardiotoxicity by alleviating oxidative damage

The possible protective effects of resveratrol (RVT) against cardiotoxicity were investigated in Wistar albino rats treated with saline, saline+doxorubicin (DOX; 20 mg/kg) or RVT (10 mg/kg)+DOX. Blood pressure and heart rate were recorded on the 1^st week and on the 7^th week, while cardiomyopathy was assessed using transthoracic echocardiography before the rats were decapitated. DOX-induced cardiotoxicity resulted in decreased blood pressure and heart rate, but lactate dehydrogenase, creatine phosphokinase, total cholesterol, triglyceride, aspartate aminotransferase and 8-OHdG levels were increased in plasma. Moreover, DOX caused a significant decrease in plasma total antioxidant capacity along with a reduction in cardiac superoxide dismutase, catalase and Na⁺,K⁺-ATPase activities and glutathione contents, while malondialdehyde, myelopreoxidase activity and the generation of reactive oxygen species were increased in the cardiac tissue. On the other hand, RVT markedly ameliorated the severity of cardiac dysfunction, while all oxidant responses were prevented; implicating that RVT may be of therapeutic use in preventing oxidative stress due to DOX toxicity.     

Effects of normalization of plasma testosterone levels in hypogonadal men on plasma levels and urinary excretion of asymmetric dimethylarginine (ADMA).

Elevated plasma levels of asymmetric dimethylarginine (ADMA) inhibit nitric oxide formation and exert a proatherogenic action. Low testosterone (T) levels are associated with increased cardiovascular risks. This study analyzed the effects of normalization of plasma T levels on plasma levels and urinary excretion of ADMA in hypgonadal men (n=10) receiving transdermal T administration. Plasma T levels, starting from clearly hypogonadal T plasma concentrations with a mean level of 4.0+/-2.72 nmol/l at baseline, rose to >10 nmol/l after 2 weeks, with plasma T levels within the normal range of men (mean level of 22.5+/-11.3 nmol/l) over the last 16 weeks of the 24 weeks of T administration. Normalization of plasma T led to a small but significant fall of plasma ADMA (519+/-55 vs. 472+/-59 nmol/l, p=0.031). The outcome of this study may be viewed as a favorable effect of normalization of plasma testosterone on plasma ADMA since even small elevations of plasma ADMA significantly increase cardiovascular risk. While this effect of normalization of plasma T may impress as favorable, most available studies on effects of T administration to hypogonadal men have not shown beneficial effects on functions of the vascular wall.     

Blood-to-retina transport of creatine via creatine transporter (CRT) at the rat inner blood-retinal barrier

The purpose of this study was to elucidate the mechanisms of blood-to-retina creatine transport across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The creatine transport across the BRB in vivo and creatine uptake in an in vitro model of the inner BRB (TR-iBRB2 cells) were examined using [(14)C]creatine. Identification and localization of the creatine transporter (CRT) were carried out by RT-PCR, western blot, and immunoperoxidase electron microscopic analyses. An in vivo intravenous administration study suggested that [(14)C]creatine is transported from the blood to the retina against the creatine concentration gradient that exists between the retina and blood. [(14)C]Creatine uptake by TR-iBRB2 cells was saturable, Na(+)- and Cl(-)-dependent and inhibited by CRT inhibitors, suggesting that CRT is involved in creatine transport at the inner BRB. RT-PCR and western blot analyses demonstrated that CRT is expressed in rat retina and TR-iBRB2 cells. Moreover, using an immunoperoxidase electron microscopic analysis, CRT immunoreactivity was found at both the luminal and abluminal membranes of the rat retinal capillary endothelial cells. In conclusion, CRT is expressed at the inner BRB and plays a role in blood-to-retina creatine transport across the inner BRB.     

The blood-cerebrospinal fluid barrier is a major pathway of cerebral creatinine clearance: involvement of transporter-mediated process

There is still incomplete evidence for the cerebral clearance of creatinine (CTN) which is an endogenous convulsant and accumulates in the brain and CSF of patients with renal failure. The purpose of this study was to clarify the transporter-mediated CTN efflux transport from the brain/CSF. In vivo data demonstrated that CTN after intracerebral administration was not significantly eliminated from the brain across the blood-brain barrier. In contrast, the elimination clearance of CTN from the CSF was 60-fold greater than that of inulin, reflecting CSF bulk flow. Even in renal failure model rats, the increasing ratio of the CTN concentration in the CSF was lower than that in the plasma, suggesting a significant role for the CSF-to-blood efflux process. The inhibitory effects of inhibitors and antisense oligonucleotides on CTN uptake by isolated choroid plexus indicated the involvement of rat organic cation transporter 3 (rOCT3) and creatine transporter (CRT) in CTN transport. rOCT3- and CRT-mediated low-affinity CTN transport with K(m) values of 47.7 and 52.0 mM, respectively. Our findings suggest that CTN is eliminated from the CSF across the blood-CSF barrier as a major pathway of cerebral CTN clearance and transporter-mediated processes are involved in the CTN transport in the choroid plexus.     

Evidence for facilitated diffusion of urea across the gill basolateral membrane of the rainbow trout (Oncorhynchus mykiss)

Recent in vivo evidence suggests that the mechanism of branchial urea excretion in the ammoniotelic rainbow trout (Oncorhynchus mykiss) is carrier-mediated. Further characterization of this proposed mechanism was achieved by using an in vitro isolated basolateral membrane vesicle (BLMV) preparation in which isolated gill membranes were used to determine a variety of physiological properties of the transporter. BLMV demonstrated two components of urea uptake, a linear component at concentrations up to 17.5 mmol x l(-1) and a saturable component (K(0.5)=0.35+/-0.01 mmol x l(-1); V(max)=0.14+/-0.02 micromol mg protein(-1) h(-1)) with a Hill constant of 1.35+/-0.18 at low, physiologically relevant urea concentrations (<2 mmol x l(-1)). Saturable uptake of urea at 1 mmol x l(-1) by BLMV was reduced by 88.5% when incubated with 0.25 mmol x l(-1) phloretin, a potent blocker of UT-type facilitated diffusion urea transport mechanisms. BLMV also demonstrated differential handling of urea versus urea analogues at 1 mmol x l(-1) concentrations and total analogue/total urea uptake ratios were 32% for acetamide and 84% for thiourea. Saturable urea uptake at 1 mmol x l(-1) was significantly reduced by almost 100% in the presence of 5 mmol x l(-1) thiourea but was not affected by 5 mmol x l(-1) acetamide or 5 mmol x l(-1) N-methylurea. Lastly, total urea uptake at 1 mmol x l(-1) by BLMV was sensitive to temperatures above and below the temperature of acclimation with a Q(10)>2 suggesting a protein carrier-mediated process. Combined, this evidence indicates that a facilitated diffusion urea transport mechanism is likely present in the basolateral membrane of the rainbow trout gill.