Distribution, metabolism and excretion of [1-14C]dolichol injected intravenously into rats.

[1-14C]Dolichol mixed in vitro with rat serum and injected intravenously into rats was rapidly cleared from the circulation in a manner consistent with a two-compartment model. About 80% of the radioactivity recovered from animals killed after 1 day was in the liver, with smaller amounts being found in lung, carcass (internal organs removed), gastrointestinal tract and contents, and spleen. The kidneys, testes and heart contained little radioactivity, and the brain did not appear to take up any [1-14C]dolichol. The half-life for the turnover of radioactivity from [1-14C]dolichol in tissues varied considerably, being 2 days for the lung, 17 for liver and about 50 days for the carcass. After 1 day, and also after 4 and 21 days, most of the radioactivity in all tissues was as [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester, although a small amount of incorporation of [1-14C]dolichol radioactivity into phospholipids was also observed. Faeces collected over the first 4 days after injection contained 13% of the [1-14C]dolichol dose, but urine and expired air contained only small amounts of radioactivity. Radioactivity in faeces was nearly all as unchanged [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester. The [1-14C]dolichol remaining in liver after 21 days appeared to be in a pool (possibly lysosomes) where most of it was not subject to excretion.     

Rates of flux through the pentose cycle in perfused rat liver. A procedure for the calculation of rates of substrate flux from 14CO2 production from [1-14C]glucose

A method for the determination of substrate flux through the pentose cycle was developed employing [1-14C]glucose in experiments with perfused rat livers. The method consists first of a kinetic analysis which differentiates between the production of 14CO2 from [1-14C]glucose via the pentose cycle and via the citrate cycle and, second of a calculation of the specific radioactivity of the hexose monophosphate pool from measured rates of glycolysis and the specific radioactivity of lactate released into the perfusate. The method was validated by experiments comparing the results of tracer infusions with [1-14C]glucose, [6-14C]glucose and [3-14C]pyruvate. In livers from fed rats perfused with 10 mM glucose, the rate of substrate flux through the pentose cycle was around 0.2 mumol X min-1 X g-1; it was about 20% of the substrate flux via glycolysis. The kinetic data were inconsistent with the existence of an L-type pentose cycle in liver.     

Tumor uptake studies of d,l-[5-14C]ornithine and d,l-2-difluoromethyl[5-14C]ornithine

The feasibility of d,l-[5-¹⁴C]ornithine ([¹⁴C]ornithine), a precursor for polyamine synthesis, and d,l-2-difluoromethyl[5-¹⁴C]ornithine ([¹⁴C]DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) were investigated for tumor localization. As an animal model, mice bearing mammary carcinoma, FM3A, were used. After i.v. injection of [¹⁴C]ornithine accumulation of radioactivity was observed in the FM3A, in which 43% of the ¹⁴C radioactivity was measured in the polyamine pool and 41% in the amino acid pool at 60 min after injection. Tumor uptake of [¹⁴C]DFMO was relatively low but constant during 60 min after injection. At 60 min after injection, 11% of the ¹⁴C was present in the acid-precipitable fraction of the FM3A, which suggests the formation of an irreversible complex of [¹⁴C]DFMO with ODC. For both compounds rapid blood clearance and high tumor-to-organ ratios were observed. Our results indicate that in connection with an enhanced polyamine synthesis in the tumors, the compounds investigated have potential as tracers for tumor detection.     

Long-term distribution and metabolism of [1-14C]dolichol injected intravenously into rats

We have previously shown that [1-14C]dolichol mixed in vitro with rat serum and injected intravenously is rapidly cleared from the circulation and appears primarily in the liver. One day after injection the liver accounted for 80% of the isotope in whole animals, whereas after 130 days it represented only 50%. During the 130 days the specific radioactivity (dpm/g liver) decreased by more than 20-fold. In contrast, the spleen retained at 130 days 85% of the radioactivity initially present and its specific radioactivity decreased by only a factor of two. At this time small amounts of isotope were also found in carcass (internal organs removed), gastrointestinal tract and contents, and lungs. Trace amounts of radioactivity were extractable from testes and kidneys, while the heart and brain were essentially free of radioactivity. At all times after injection nearly all the radioactivity present in all tissues was still associated with dolichol. Only trace amounts of [1-14C]dolichyl fatty acyl ester and no [1-14C]phosphorylated derivatives of dolichol were present in the liver and spleen removed 156 days postinjection. Fractionation of liver between 1h and 93 days after injection suggested that [1-14C]dolichol becomes associated primarily with a lysosome-enriched fraction. The accumulation of [1-14C]dolichol in this and other subcellular compartments involved both an inward and outward flow of radioactivity, suggesting that deposition of dolichol in lysosomes is not a one-way terminal process.     

Elimination of low steady-state concentrations of E15,6-3H2]prostaglandin E1 in the pulmonary and the systemic circulations of anaesthetized rats

The elimination of [3H]prostaglandin E1 in anaesthetized rats was studied by continuous intravenous or intraarterial infusions, producing steady-state concentrations at the level of endogenous prostaglandin E2 in mixed venous blood. Blood samples (0.5 ml) were collected from the carotid artery or the right atrium, respectively. The levels of [3H]prostaglandin E1 were measured at different infusion time intervals and the 3H-labeled hydrophobic metabolites characterized. Cardiac output was estimated by a modification of the dye injection method, using 125I-labelled albumin as the marker. From the cardiac output and the rate of infusion, the fractional clearance of the lung and the systemic beds in the steady-state situation were estimated to 88.3 +/- 3.2% and 54.1 +/- 15.2% (mean +/- S.D.), RESPECTIVELY. The hydrophobic metabolites were characterized chromatographically on Sephadez LH-20 columns, using synthetically prepared [14C]prostaglandin metabolites as internal standards and markers. The identities of some metabolites were further established by derivative formation to a constant [3H]/[14C] ratio. The major metabolite was 15-keto-13,14-dihydro-[3H]prostaglandin E1, while 15-keto-[3H]prostaglandin E1 and 13,14-dihydro-[3H]prostaglandin E1 could not be demonstrated.     

Preparation of bromo[1-14C]acetyl-coenzyme A as an affinity label for acetyl-coenzyme A binding sites

Bromo[1-14C]acetyl-CoA has been prepared from CoASH and the N-hydroxysuccinimide ester of bromo[1-14C]acetic acid, and unlabeled bromoacetyl-CoA by reaction of CoASH with bromoacetyl bromide. The products were purified by high-pressure liquid chromatography. Purified bromoacetyl-CoA was characterized, and found to be a potent alkylating agent with a substantial stability in aqueous solution: it decomposed at 30 degrees C and pH 6.6 and 8.0 with halftimes of 3.3 and 2.5 h, respectively. The major breakdown products were CoASH and CoAS X CO X CH2 X SCoA. Bromo[1-14C]acetyl-CoA has been used to affinity label the acetyl-CoA binding site of 3-hydroxy-3-methylglutaryl-CoA synthase from ox liver. It was found to irreversibly inhibit the enzyme activity and bind covalently with a stoichiometry for complete inhibition of about 0.8 mol/mol enzyme dimer.     

A preparation and purification of [1-14C]acetylcarnitine.

[1-14C]Acetylcarnitine was prepared from [1-14C]acetate and L-carnitine using acetyl-CoA synthetase and carnitine acetyltransferase. The product was purified by ion-exchange and thin-layer chromatography. Conversion of [1-14C]acetate to [1-14C]acetylcarnitine was better than 90% and overall recovery of the pure product was greater that 80%.     

Kinetics and organ distribution of [14C]-sialic acid-GM3 and [3H]-sphingosine-GM1 after intravenous injection in rats.

Serum kinetics and organ distribution of [14C]-sialic acid-GM3 and [3H]-sphingosine-GM1, administered as an intravenous bolus, were analysed in Wistar rats. [3H]-GM1 and [14C]-GM3 had serum half-lives of 1.4 hours and 1.8 hours, respectively. Three hours after injection 75% of the GM1- and 38% of the GM3-associated label were present in the liver. Smaller yet significant amounts of label were present in the central nervous system, kidneys and lung. In vitro studies showed that [14C]-GM3 and [3H]-GM1 incubated with serum were predominantly bound to the High Density Lipoprotein and the Low Density Lipoprotein fractions. These results suggest a rapid serum clearance of exogenous gangliosides by the liver in rats.     

The metabolism of (1-14C) stearic acid in rat testicular tissue.

(1) The metabolism of stearic acid was studied in vivo following intratesticular injection of [1-14C] stearate. Soon after injection 14C activity was found mainly in the free fatty acid pool. This was followed at later time periods by transfer of label primarily to the phosphatide pool. During each time period significant amounts of label were recovered at 14CO2. (2) Analysis of 14C-labeled fatty acids from the injected testes demonstrated an initial rapid rate of oxidation and desaturation of [1-14C] stearate followed by a slower steady state rate. It was concluded that the initial rate was due to the rapid turnover of the highly labeled free fatty acid pool followed by a much slower rate as [14C] stearate was esterified to the more metabolically stable phospholipids. Elongation of the labeled stearic or its desaturated derivative was not observed. (3) The rate of desaturation in vitro of stearic acid was measured in microsomal preparations from rat testes and found to be 12.0 +/- 0.5 pmol/min/mg compared to the estimated in vivo value of 22 pmol/min/mg and the value of 390 pmol/min/mg for hepatic microsomal desaturase.     

Effect of insulin and cortisol on oxidation of (1-14C)glucose, (6-14C)glucose, (1-14C)palmitate and (1-14C)leucine in the tissues of swine during the neonatal period

The intensity of [1-14C]glucose, [6-14C]glucose, [1-14C]palmitate and [1-14C]leucine oxidation and the effect of insulin and hydrocortisone on this process were studied in the brain, duodenum mucosa, liver and skeletal muscle of 1- and 5-day old piglets in vitro. Most of the studied substrates are oxidized in the tissues of 5-day piglets more intensively. Insulin stimulates oxidation of [1-14C]glucose, [6-14C]glucose and [1-14C]leucine in the brain and duodenum mucosa in 1- and 5-day old piglets, while in the liver and skeletal muscle--only in 5-day old piglets. Hydrocortisone administration enhances oxidation of [1-14C]leucine in most of the studied tissues in 1-day piglets and oxidation of [1-14C]glucose and [6-14C]glucose--in 5-day piglets. Both hormones produce no essential influence on the intensity of [1-14C]palmitate oxidation in the studied tissues of piglets or somewhat weaken it.     

Gluconeogenesis from acetone in starved rats.

To non-anaesthetized rats starved for 3 days, [U-14C]acetone, NaH14CO3, L-[U-14C]lactate, [2-14C]acetate or D-[U-14C]- plus D-[3-3H]-glucose was injected intravenously. From the change in the plasma concentration of labelled acetone versus time after the injection, the metabolic clearance rate of acetone was calculated as 2.25 ml/min per kg body wt., and its rate of turnover as 0.74 mumol/min per kg. The extent and time course of the labelling of plasma glucose, lactate, urea and acetoacetate were followed and compared with those observed after the injection of labelled lactate, acetate and NaHCO3. The labelling of plasma lactate was rapid and extensive. Some 1.37% of the 14C atoms of circulating glucose originated from plasma acetone, compared with 44% originating from lactate. By deconvolution of the Unit Impulse Response Function of glucose, it was shown that the flux of C atoms from acetone to glucose reached a peak at about 100 min after injection of labelled acetone. In comparable experiments the transfer from lactate reached a peak at 14 min after the injection of labelled lactate. It was concluded that acetone is converted into lactate to a degree sufficient to account for the labelling of plasma glucose and is thus a true, albeit minor, substrate of glucose synthesis in starved rats.     

Monitoring flux through the oxidative pentose phosphate pathway using [1-14C]gluconate

The aim of this work was to examine the metabolism of exogenous gluconate by a 4-day-old cell suspension culture of Arabidopsis thaliana (L.) Heynh. Release of (14)CO(2) from [1-(14)C]gluconate was dependent on the concentration in the medium and could be resolved into a substrate-saturable component (apparent K(m) of approximately 0.4 mM) and an unsaturable component. At an external concentration of 0.3 mM, the rate of decarboxylation of applied gluconate was 0.2% of the rate of oxygen consumption by the cells. There was no effect of 0.3 mM gluconate on the rate of oxygen consumption, or on the rate of (14)CO(2) release from either [1-(14)C]glucose or [6-(14)C]glucose by the culture. The following observations argue that gluconate taken up by the cells is metabolised by direct phosphorylation to 6-phosphogluconate and subsequent decarboxylation through 6-phosphogluconate dehydrogenase. First, more than 95% of the label released from [1-(14)C]gluconate during metabolism by the cell culture was recovered as (14)CO(2). Secondly, inhibition of the oxidative pentose phosphate pathway (OPPP) by treatment with 6-aminonicotinamide preferentially inhibited release of (14)CO(2) from [1-(14)C]gluconate relative to that from [1-(14)C]glucose. Thirdly, perturbation of glucose metabolism by glucosamine did not affect (14)CO(2) from [1-(14)C]gluconate. Fourth, stimulation of the OPPP by phenazine methosulphate stimulated release of (14)CO(2) from [1-(14)C]gluconate to a far greater extent than that from [1-(14)C]glucose. It is proposed that measurement of (14)CO(2) from [1-(14)C]gluconate provides a simple and sensitive technique for monitoring flux through the OPPP pathway in plants.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clearance and tissue distribution of functionalized polymeric liposomes from the blood stream of rats

Polymeric liposomes containing a synthetic porphinato-iron-imidazole complex (hemoglobin or red blood cell model) were labeled by introducing 1,2-di[1-14C]palmitoyl-sn-glycero-3-phosphocholine into their polymerized bilayers. After intravenous injection into rats, their clearance from a blood stream was measured. The apparent half-life time (50% disappearance time) was about 14 +/- 2 h. Their tissue distribution was determined with time by whole autoradiographic measurement.     

The effect of etomoxir on glucose turnover and recycling with [1-14C], [3-3H]-glucose tracer in pigs

Infusion of etomoxir to 4 fasted pigs caused significant (48%) falls in blood glucose concentrations. To assess whether inhibition of hepatic glucose production or increase of peripheral glucose utilisation is causally associated, a primed infusion of [3-3H]-glucose and [1-14C]-glucose was used, and glucose turnover rates, recycling and metabolic clearance rate of glucose were determined. No effects of infusion of etomoxir on glucose turnover rates could be found. Recycling of glucose carbon was affected to a relatively small extent. The metabolic clearance rate, however, increased by 126% from 5.0 +/- 0.7 ml/kg x min in the control group to 11.3 +/- 3.5 ml/kg x min in the treated group (mean +/- SEM; P less than 0.05). We conclude that under fasting conditions an increase in glucose utilization plays a major part in the blood glucose lowering effect of etomoxir.     

Effect of insulin on the metabolic distribution of carbons 1, 2, and 3 of pyruvate

It has long been known that the carbons of pyruvate are converted to CO2 at different points in the metabolic process. This report deals with the observation that insulin affects the oxidation of carbons 2 and 3 primarily and has little effect on the oxidation of the carboxyl carbon. Oxidation of different carbons of pyruvate and their incorporation into various metabolic components was studied in isolated rat hepatocytes. Insulin stimulated the 14CO2 production from [2-14C]- and [3-14C]pyruvate and from [U-14C]alanine. However, it had little or no effect on the activity of the pyruvate dehydrogenase complex as measured by the evolution of 14CO2 from [1-14C]pyruvate or [1-14C] alanine. Insulin also stimulated the incorporation of carbons 2 and 3 of pyruvate into protein but had no effect on the incorporation of carbon 1. Incorporation of [1-14C]- and [U-14C]alanine into protein was differentially enhanced by insulin in a manner similar to that of the pyruvate carbons. The fact that insulin stimulates the incorporation of [1-14C]alanine into protein but not [1-14C]pyruvate suggests the possibility of a compartmentation of pyruvate metabolism in the isolated hepatocytes. These studies show that the stimulation of [2-14C]- and [3-14C]pyruvate incorporation into protein involves the stimulatory effect of insulin on the activity of the Krebs cycle which is evident from the fact that insulin did not stimulate the pyruvate carbons to enter protein via alanine but the incorporation via glutamate was increased by about 40%.     

Long-chain acyl-coenzyme A synthetase from rat brain microsomes. Kinetic studies using [1-14C]docosahexaenoic acid substrate

The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexaenoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 degrees C. The apparent Km values for ATP (185 microM), CoA (4.88 microM), MgCl2 (555 microM) and [1-14C]docosahexaenoic acid (26 microM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 degrees C (t1/2:3.3 min) and 37 degrees C (t1/2:26.5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 degrees C with the activation energies of 74 kJ/mol between 0 degrees C and 27 degrees C and 30 kJ/mol between 27 degrees C and 45 degrees C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 microM and 29 microM respectively, with V values of 13 and 46 nmol X min-1 X mg protein-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 degrees C and 28 degrees C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 microM and 19 microM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the competition of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct measurement of the lumped constant for 2-deoxy-[1-(14)C]glucose in vivo in human skeletal muscle

The lumped constant (LC) is used to convert the clearance rate of 2-deoxy-D-glucose (2-DG(CR)) to that of glucose (Glc(CR)). There are currently no data to validate the widely used assumption of an LC of 1.0 for human skeletal muscle. We determined the LC for 2-deoxy-[1-(14)C]glucose (2-DG) in 18 normal male subjects (age, 29+/- 2 yr; body mass index, 24.8+/-0.8 kg/m(2)) after an overnight fast and during physiological (1 mU x kg(-1) x min(-1) insulin infusion for 180 min) and supraphysiological (5 mU x kg(-1) x min(-1) insulin infusion for 180 min) hyperinsulinemic conditions. Normoglycemia was maintained with the euglycemic clamp technique. The LC was measured directly with the use of a novel triple tracer-based method. [3-(3)H]glucose, 2-[1-(14)C]DG, and [(12)C]mannitol (Man) were injected as a bolus into the brachial artery. The concentrations of [3-(3)H]glucose and 2-[1-(14)C]DG (dpm/ml plasma) and of Man (micromol/l) were determined in 50 blood samples withdrawn from the ipsilateral deep forearm vein over 15 min after the bolus injection. The LC was calculated by a formula involving blood flow calculated from Man and the Glc(CR) and 2-DG(CR). The LC averaged 1.26+/-0.08 (range 1.06-1.43), 1.15+/-0.05 (0.99-1.39), and 1.18+/-0.05 (0.97-1.37) under fasting conditions and during the 1 and 5 mU x kg(-1). min(-1) insulin infusions (not significant between the different insulin concentrations, mean LC = 1.2, P<0.01 vs. 1.0). We conclude that, in normal subjects, the LC for 2-DG in human skeletal muscle is constant over a wide range of insulin concentrations and averages 1. 2.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

Carrier-Mediated Transport of Chloride Across the Blood-Brain Barrier

36Cl concentrations in each of eight brain regions and in cisternal cerebrospinal fluid (CSF) were determined 30 min after the intravenous injection of 36Cl in dialyzed-nephrectomized rats with plasma Cl concentrations between 14 and 120 mumol X ml-1. CSF 36Cl exceeded 36Cl concentrations in brain extracellular fluid. The calculated blood-to-brain transfer constants for Cl, kCl, ranged from 1.8 X 10(-5) S-1 at the parietal cortex to 3.8 X 10(-5) S-1 at the thalamus-hypothalamus. kCl fell by 42-62% when mean plasma [Cl] was elevated from 16 to 114 mumol X ml-1. Brain uptake of [14C]mannitol or of 22Na was independent of plasma [Cl], but 22Na influx into CSF fell when plasma [Cl] was reduced. Cl flux into brain and CSF could be represented by Michaelis-Menten saturation kinetics, where, for the parietal cortex, Km = 43 mumol X ml-1 and Vmax = 2.5 X 10(-3) mumol X S-1 X g-1, and for CSF Km = 68 mumol X ml-1. At least 80% of 36Cl influx into the parietal cortex was calculated to occur at the cerebrovascular endothelium, whereas the remainder was derived from tracer that first entered CSF. The CSF contribution was greater at brain regions adjacent to cerebral ventricles. The results show that Cl transport at the cerebrovascular endothelium as well as at the choroid plexus epithelium is a saturable concentration-dependent process, and that the CSF is a significant intermediate pathway for Cl passage from blood to brain.