Maximal Transport Capacity Research Articles

Glucocorticoids Inhibit Intestinal Phosphate Absorption in Developing Rabbits

Children are apt to develop osteoporosis during chronic glucocorticoid therapy, in part due to suppression of intestinal calcium absorption. Little is known about effects of glucocorticoids on phosphate absorption. To characterize effects of glucocorticoids on intestinal phosphate transport, 4-wk-old suckling rabbits were injected with methylprednisolone. Twenty-four hours following injection, phosphate transport was studied in brush border membrane vesicles. Vesicles from steroid-treated and control animals demonstrated sodium-phosphate cotransport. Uptake was significantly greater in controls than in methylprednisolone-treated animals. Controls demonstrated an “overshoot” in that peak phosphate uptake exceeded equilibrium by nearly 100%, whereas minimal overshoot was observed in methylprednisolone-treated animals. Vmax and Km for sodium-dependent phosphate transport were 556.6 ± 13.2 pmol-mg protein-1·10 s-1 and 0.051 ± 0.01 mmol/L, respectively, for controls and 285.4 ± 3.5 pmol·mg protein-1·10 s-1 and 0.044 ± 0.01 mmol/L for methylprednisolone-treated animals. Although phosphate uptake was greater in controls than in methylprednisolone-treated animals, maximal stimulation of phosphate uptake occurred between 75 and 100 mmol/L sodium in both groups. Uptake was half-maximally stimulated by 32.5 ± 8.1 mmol/L sodium in controls and 41.4 ± 6.2 mmol/L sodium in treated animals. These results indicate that within 24 h methylprednisolone suppressed intestinal sodium-coupled phosphate transport. This is due to decreased maximal transport capacity without changes in transporter affinity for phosphate or sodium.

Ontogeny of intestinal phosphate absorption in rabbits.

The ontogeny of intestinal phosphate transport was examined in brush-border membrane vesicles prepared from 2-wk, 4-wk, 6-wk, and 3-mo-old rabbits. At all four ages, vesicles prepared from the duodenum demonstrated sodium-phosphate cotransport in that uptake in the presence of a sodium gradient was significantly greater than uptake in the presence of a potassium gradient. While sodium-independent phosphate uptake was comparable at all ages, sodium-phosphate cotransport was greatest in the youngest animals and declined with increasing age. Peak phosphate uptake was 10-fold the equilibrium value at 2 wk and declined to 3-fold in adults. Maximal transport capacity of sodium-phosphate cotransport fell from 2,292 +/- 161 at 2 wk to 286 +/- 12 pmol.mg-1.10 s-1 at 3 mo, whereas the Michaelis constant did not change with age, varying between 0.032 and 0.054 mM. At all ages, uptake was half-maximally stimulated between 30 and 50 mM sodium, and Hill coefficients were between 1.5 and 2. Generation of inside negative diffusion potentials did not significantly enhance phosphate uptake at any age. These data suggest that throughout development, the transport of each phosphate molecule across the rabbit duodenal brush-border membrane is coupled to the transport of two sodium molecules. There were distinct developmental changes in the distribution of the sodium-phosphate cotransport along the length of intestine. In 2-wk old animals, sodium-phosphate cotransport was present in the duodenum, proximal and distal jejunum, and proximal ileum. In 4-wk-old animals, sodium-phosphate cotransport was present throughout the duodenum and proximal and distal jejunum, and in 6-wk and 12-wk-old animals, sodium-phosphate cotransport was observed only in the duodenum and proximal jejunum.

The rabbit jejunal ‘imino carrier’ and the ileal ‘imino acid carrier’ describe the same epithelial function

For rabbit jejunal brush border vesicles an ‘imino carrier’ has been defined as a sodium-depedent, alanine-resistant transporter of cyclic imino acids, while for intact rabbit jejunal and ileal epithelia an ‘imino acid carrier’ has been defined as a sodium-dependent transporter of both aliphatic and cyclic imino acids. This study on intact rabbit intestine examines whether these two terms describe the same epithelial function. The K 1 2 Pro and the K i Pro against J mc MeAIB are identical and so are K 1 2 MeAIB and K i MeAIB against J mc Pro. Likewise, K i Leu is tha same against the transport of both proline and MeAIB. Is is, therefore, concluded that the terms ‘imino carrier’ and ‘imino acid carrier’ describe the same epithelial function: A sodium-dependent, relatively high afinity, saturable transporter of both aliphatic and cyclic imino acids. Estimates of the apparent affinity and inhibitory constants for MeAIB, proline and leucine confirm that the jejuno-ileal variation of amino acid transport along the rabbit small intestine is a variation of maximal transport capacity.

Altered glucose carrier expression: mechanism of intestinal adaptation during streptozocin-induced diabetes in rats

Intestinal adaptation of glucose transport during streptozocin-induced diabetes in rats was examined using microdensitometric analysis of [3H]phlorizin binding. Results of specific phlorizin binding were correlated with measurements of maximal transport capacity, carrier affinity, villus height, and enterocyte birth rate determined by the metaphase arrest technique. Animals diabetic for 14 days (acute) and 60 days (chronic) were compared with age-matched controls. In the jejunum, adaptation occurred only in chronically diabetic rats and consisted of a 10-fold increase in the density of phlorizin binding sites in the upper villus region (i.e., that portion normally transporting glucose), while in the ileum, adaptation occurred both in acute and chronically diabetic rats and consisted of 1) a 3-fold increase in density of phlorizin binding sites in the upper villus region of acutely diabetic rats and 2) an increased density in the upper villus region as well as the recruitment of phlorizin binding sites in the mid to lower villus region (i.e., that portion not normally transporting glucose) of chronically diabetic rats. Enhancement of glucose Vmax and villus length accompanied changes in binding, whereas enterocyte birth rates were similar in each group.

Transient experimental investigation of micro heat pipes

An experimental investigation was conducted on several small, tapered micro heat pipes specifically designed for use in the thermal control of ceramic chip carriers to verify the operation, measure the performance limits and transient behavior, and determine the accuracy of a previously developed numerical model. Several heat pipes were evaluated for transient conditions, i.e., startup or rapid changes in the thermal load. The experimental data are compared with the results of a previously developed analytical model to determine the accuracy of the model and verify the predicted trends. The experimental results indicated that the transient numerical model is capable of accurately predicting the maximum transport capacity prior to the onset of dry out, the temperature distribution throughout the longitudinal position, and the temperature difference between axial locations on the heat pipe to within 0.3°C. Although the numerical model was found to accurately predict the steady-state behavior, the numerical model substantially underestimated the transient response.

Adaptation of intestinal glucose transport in rats with diabetes mellitus occurs independent of hyperphagia

Chronic diabetes enhances intestinal absorption of glucose and induces hyperphagia. We examined the enhanced intestinal absorption of glucose in ad libitum-fed rats with streptozocin-induced diabetes mellitus and compared these results with those obtained from pair-fed diabetic animals. Maximal transport capacity (Vmax) and carrier affinity (K0.5) were determined by measuring jejunal and ileal short circuit current (Isc) responses to varying concentrations of 3-O-methyl-D-glucopyranose and D-glucose. Pair-fed diabetic animals maintained the same body weight as animals fed ad libitum, although ad libitum-fed diabetic rats had an increased oral chow intake. Age-matched control rats maintained a constant jejunal and ileal Vmax and K0.5 throughout the study. Diabetic rats fed ad libitum demonstrated an enhanced Vmax and K0.5 in both jejunum and ileum. Pair feeding diabetic animals further enhanced jejunal Vmax while lowering jejunal K0.5 levels. In contrast, pair feeding diabetic animals delayed and blunted changes in ileal Vmax and prevented changes in ileal K0.5. In conclusion, signals other than those of hyperphagia regulate kinetic changes in glucose absorption during diabetes mellitus. Furthermore, these changes have differing effects on jejunum and ileum.

Dose-Dependent Pharmacokinetics of Enprofylline and Its Renal Handling in Rats

The effect of dosage on the pharmacokinetics of the potent bronchodilator enprofylline (3-propylxanthine; PX) and its renal handling were investigated in rats. Enprofylline (PX) was administered iv in dosages of 2.5, 10, 20, and 40 mg/kg, and PX concentration in plasma and urine was determined by HPLC. The pharmacokinetic parameters were estimated by model-independent methods. The disappearance of PX from plasma was delayed as dosage was increased. The corresponding pharmacokinetic parameters also showed dose dependency; increases in the volume of distribution (Vd) and mean residence time (MRT) and a decrease in total body clearance (CLT) were observed as dosage was increased from 2.5 to 40 mg/kg. Approximately 80% of the dose, however, was excreted in urine as unchanged PX. Plasma protein binding studies of PX showed concentration dependency and allowed determination of binding parameters, with an apparent dissociation constant (Kd) of 162.50 μM and a binding capacity (nP) of 565.23 μM. Some pharmacokinetic parameters for unbound PX calculated by total plasma concentration and binding parameters also showed dose-dependent characteristics. However, no significant change in Vd for unbound PX was observed among administered doses, indicating that the distribution of PX into the body tissues is not changed by an increase in dosage. Renal clearance of unbound PX significantly increased as plasma concentration decreased. The maximum transport capacity (Vmax) and the Michaelis-Menten constant (Km) for tubular secretion were 60.53 μg/min and 2.27 μg/mL, respectively. The aim of the present study is to demonstrate that both saturable tubular secretion and concentration-dependent protein binding are responsible for the dose- dependent pharmacokinetics of PX in rats.

Palmitate binding to and efflux kinetics from human erythrocyte ghost

At 0°C, pH 7.3, palmitate (PA) binds to human erythrocyte ghosts suspended in 0.2% bovine serum albumin (BSA) solution with molar ratios of PA to BSA, ν, between 0.2 and 1.3. The binding depends on the water phase PA concentration, measured in equilibrium experiments, using BSA-filled ghosts as semipermeable bags. The saturable binding has a capacity of 19.4 ± 7.5nmol g −1 packed ghosts (7.2 · 10 9 cells) and K d = 13.5 ± 5nM. PA exchange efflux kinetics to 0.2% BSA is recorded from ghosts without and with 0.2% BSA with a resolution time of about 1 s. Data are analyzed in terms of compartmental models. Using BSA-free ghosts the kinetics is essentially monoexponential. The rate constant is 0.0287 ± 0.0022s −1. Using ghosts with BSA, the kinetics is biexponential with widely different rate constants. Extrapolated zero-time values reflect, according to additional investigations, ‘instantaneous’ release of PA from the outer surface of the ghosts. Analyses of the biexponential curve up to about 55% tracer efflux assign unequivocally values to three model parameters. (1) k 1, the dissociation rate constant of the PA-BSA complex is(1.47 ± 0.03) · 10 −3 s −1 and (2.56 ± 0.08) · 10 −3 s −1 and (4.08 ± 0.13) · 10 −3 s −1 at ν = 0.2, 0.6 and 1.4, respectively. (2) k 3 *, the overall rate constant of PA transport from the inside of the ghost membrane to the medium is 0.0269 ± 0.0020 s −1 independent of ν. (3) Q kin, the ratio of PA on the inside of the membrane to PA on BSA within the ghosts is ν dependent and smaller than a corresponding ratio Q eq measured in equilibrium by a value corresponding to PA on the outer surface. This fraction is released with a rate constant, k 5, which is of the order of 1 s −1. The data suggest a maximum PA transport capacity, J max, of 2 pmol min −1 cm −2, 0°C, pH 7.3.

Evidence for altered Na+/H+ antiport activity in cultured skeletal muscle cells and vascular smooth muscle cells from the spontaneously hypertensive rat

1. Intracellular pH and Na+/H+ antiport activity were determined by a fluorimetric method in cultured skeletal muscle cells (myoblasts) and aortic vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats. 2. The intracellular pH was significantly more alkaline at three different extracellular pH values in both myoblasts and vascular smooth muscle cells from the spontaneously hypertensive rats than in those from the normotensive control rats. 3. A kinetic analysis of the Na+/H+ antiport activity in these cells showed that the raised activity in the spontaneously hypertensive rats was due to an increased maximal transport capacity in vascular smooth muscle cells and to an increase in the affinity of the antiport for internal H+ in the myoblasts. 4. When the extracellular pH was reduced in the skeletal muscle cells of both types of rat, the intracellular pH fell. However, in vascular smooth muscle cells, a reduction in the extracellular pH was not associated with a fall in the intracellular pH. This resistance of the intracellular pH to changes in the extracellular pH differentiates vascular smooth muscle cells from other cells that have been studied in this way.

Dose-Dependent Pharmacokinetics of 3′-azido-2′,3′-dideoxyuridine in Rats

The pharmacokinetics of 3′-azido-2′,3′-dideoxyuridine (AzddU; CS-87), an inhibitor of human immunodeficiency virus (HIV) replication in vitro, were characterized in rats. AzddU was administered intravenously at doses of 10, 50, 100 and 250mgkg−1. Plasma and urine AzddU concentrations were measured by HPLC. Plasma AzddU concentrations declined in a biexponential fashion with a terminal half-life of approximately 1.5h. The disposition of AzddU was independent of dose over the dosage range of 10–100mgkg−1; however, the pharmacokinetics of the nucleoside exhibited non-linearities after 250mgkg−1. Over the dose range of 10–100mgkg−1 AzddU, total clearance and renal clearance averaged 2.13lh−1kg−1 and 1.46lh−1kg−1, respectively. Total clearance was significantly lower after 250mgkg−1 ( CIT = 1.32lh−1kg−1) owing to a decreased renal clearance ( CIR = 0.69lh−1kg−1) of AzddU. Renal clearance exceeded glomerular filtration rate, indicating that active renal tubular secretion was involved in the renal excretion of the compound. The maximum transport capacity ( Tmax) and the Michaelis–Menton constant ( Km) for the tubular secretion mechanism were 142.2mg h−1 and 60.4mg l−1, respectively. The high values for Tmax and Km explain the high renal clearance of AzddU and the linearity of renal excretion over a wide range of drug concentrations. However, at very high AzddU concentrations active tubular secretion is saturable. Nonrenal clearance was independent of dose with a mean value of 0.66lh−1kg−1. Steady-state volume of distribution was similar at all doses averaging 1.05lkg−1. Thus, the disposition of AzddU is linear over the dose range of 10–100mgkg−1, but becomes dose dependent with decreases in renal and total clearances after 250mgkg−1 AzddU.

Active absorption of conjugated bile acids in vivo: Kinetic parameters and molecular specificity of the ileal transport system in the rat

Active transport of conjugated bile acids by the distal ileum is required for efficient enterohepatic cycling of bile acids. Experiments were performed in the rat to obtain accurate values for Tmax and Michaelis constant (Km) of the absorptive area of the rat ileum and to define the structural specificity of the transport system. The distal fifth (20 cm) of the small intestine from an anesthetized animal with a biliary fistula was perfused using solutions of 10 taurine-conjugated bile acids; a flow rate was used that was sufficiently high such that unstirred water layer effects were negligible and the intraluminal concentration remained unchanged throughout the perfused segment. The absorption rate was equated with the rate of hepatic bile acid secretion. Values of Tmax (μmol/min · kg) were markedly influenced by bile acid structure: cholyltaurine, 12.9; ursocholyltaurine, 9.6; ursodeoxycholyl taurine, 5.0; and lagodeoxycholyl-(3α, 12β-dihydroxy-cholanoic acid)-taurine, 1.2. Decreasing the length of the side chain of ursodeoxycholate conjugates from 8 to 6 carbon atoms was associated with a modest increase in Tmax values from 5.0 to 9.1 μmol/min · kg. Values of Km correlated with Tmax values and ranged from 0.5 to 5 mmol/L, being highest for those bile acids that were best transported. The Tmax for cholyltaurine transport was not reached when the intraluminal concentration was as high as its critical micellization concentration, precluding the definition of its Tmax; however, for ursocholyltaurine, with a critical micellization concentration of 40 mmol/L, saturation of transport was clearly shown. Kinetic parameters could not be obtained for two common dihydroxy conjugates (chenodeoxycholyltaurine and deoxycholyltaurine) because at a transport rate of 2 μmol/ min · kg systemic toxicity and death occurred. These studies define the maximal transport capacity of the rat ileum for taurine-conjugated bile acids; they indicate that the ileal transport system in the rat is of low affinity and high capacity for taurine conjugates of hydrophilic bile acids, and they show that both nuclear substituants and side chain length influence the transport rate of taurine-conjugated bile acids.

Effects of malnutrition on microvillus membrane glucose transport and physical properties

We examined sodium-dependent glucose transport, membrane lipid composition, and membrane fluidity in microvillus membrane vesicles isolated from the distal intestine of infant rabbits subjected to protein-energy malnutrition and age-matched controls. In vesicles from malnourished animals, sodium-dependent glucose transport was significantly enhanced, as evidenced by a twofold increase in maximal transport capacity, Jmax. Carrier affinity for glucose, as assessed by the Km of the transport process, was unaffected. These alternations were associated with marked changes in microvillus membrane composition. Malnourished animals had an increase in the lipid-to-protein ratio of the microvillus membrane, which suggests that malnutrition might be associated with either a reduction in membrane protein or an increase in membrane lipid. This would be expected to increase the fluidity of the microvillus membrane. However, we observed no differences in either the static or dynamic component of membrane fluidity, using multiple fluorescent probes, between dietary groups. Further analysis of membrane lipids was undertaken to establish whether quantitative differences in lipid subclasses could explain this discrepancy. We found that nutrient deprivation produced numerous alterations in membrane lipids. The major findings were an increase in both the cholesterol-to-phospholipid and phosphatidylethanolamine-to-phosphatidylcholine ratios. Both alterations would be expected to decrease membrane fluidity and presumably represent a compensatory response to the loss of membrane protein. Thus chronic postnatal protein-energy malnutrition initiates several adaptive responses that include major alterations in the chemical composition of the microvillus membrane. The resulting effect preserves efficient glucose transport and maintains the physical properties of the microvillus membrane.

Interaction of fluoxetine with the human placental serotonin transporter

The interaction of fluoxetine, a non-tricyclic antidepressant, with the human placental serotonin transporter was investigated by studying its influence on [ 3H]paroxetine binding to the transporter and on [ 3H]serotonin uptake via the transporter. These studies were done using brushborder membrane vesicles purified from normal term human placentas. Fluoxetine inhibited binding of paroxetine to the membrane vesicles in a concentration-dependent manner, with a K i value of 3 nM. Kinetic analysis revealed that the inhibition was competitive because the presence of 10 nM fluoxetine increased the k d for paroxetine from 72 to 461 pM, but had no effect on the B max . Fluoxetine also caused a time-dependent dissociation of paroxetine already bound to the transporter. The dissociation followed first-order kinetics. Uptake of serotonin in these membrane vesicles was also inhibited by fluoxetine. The inhibition was concentration dependent with a K i value of 66 nM at pH 7.5 and 80 nM at pH 6.5. The effect of fluoxetine on the uptake kinetics was to increase the apparent dissociation constant ( K l ) for serotonin without influencing the maximal transport capacity ( V max ). The results demonstrate that fluoxetine is a high-affinity ligand and a potent inhibitor of the serotonin transporter found in the human placental brush-border membrane.

Steady-State Modeling and Testing of a Micro Heat Pipe

A combined experimental and analytical investigation was conducted to identify and understand better the phenomena that govern the performance limitations and operating characteristics of micro heat pipes—heat pipes so small that the mean curvature of the vapor—liquid interface is comparable in magnitude to the reciprocal of the hydraulic radius of the flow channel. The analytical portion of the investigation began with the development of a steady-state model in which the effects of the extremely small characteristic dimensions on the conventional steady-state heat pipe modeling techniques were examined. In the experimental portion of the investigation, two micro heat pipes, one copper and one silver, 1 mm2 in cross-sectional area and 57 mm in length, were evaluated experimentally to determine the accuracy of the steady-state model and to provide verification of the micro heat pipe concept. Tests were conducted in a vacuum environment to eliminate conduction and convection losses. The steady-state experimental results obtained were compared with the analytical model and were found to predict accurately the experimentally determined maximum heat transport capacity for an operating temperature range of 40° C to 60° C. A detailed description of the methodology used in the development of the steady-state model along with a comparison of the predicted and experimental results are presented.

Kinetics of the human leucocyte N a + - H + antiport in essential hypertension

Many membrane abnormalities have been described in human essential hypertension that may lead to an increased intracellular Na+ content, an example being reduced Na+ efflux by the sodium pump. We have previously found increased Na(+)-H+ antiport activity in leucocytes of hypertensive subjects. In the present study we examined the kinetics of this pump in 16 hypertensive and 20 carefully matched normotensive subjects by loading cells to different intracellular pH levels (as measured by fluorimetry) using a double-ionophore technique. The maximal rate of ethyl isopropyl amiloride-sensitive H+ efflux was significantly raised in leucocytes from the hypertensive subjects [75.3 +/- 6.2 versus 48.8 +/- 2.1 mmol/l per min in normotensives (mean +/- s.e.m.); P less than 0.001]. There was no difference in the affinity of the Na(+)-H+ antiport for intracellular H+. Intracellular buffering power at different internal pH levels in the range 6.0-7.1 did not differ in the two groups. We conclude that one reason for the reported intracellular alkalinity and increased sodium content of leucocytes from hypertensive subjects in bicarbonate-free media could be an increased number of active Na(+)-H+ exchangers or an increased turnover rate for each exchanger. A similar defect in vascular smooth muscle could account for the increased tone and thickening of the media. The abnormal maximal transport capacity of the leucocyte may be a useful membrane marker for future studies in human hypertension.

Leucocyte Na+/H+ antiport activity in Type 1 (insulin-dependent) diabetic patients with nephropathy

The development of proteinuria in Type 1 (insulin-dependent) diabetic patients may depend on predisposition to essential hypertension in addition to poor glycaemic control. Previous work has shown increased leucocyte Na+/H+ antiport activity in essential hypertension and increased erythrocyte Li+/Na+ exchange in Type 1 diabetic patients with proteinuria. To test whether susceptibility to nephropathy in Type 1 diabetes was linked to abnormalities of leucocyte Na+/H+ antiport activity, we measured the intracellular pH and kinetics of the Na+/H+ antiport in 19 Type 1 diabetic subjects with, and 15 diabetic subjects without albuminuria and compared them to 25 matched normal control subjects. Intracellular pH (mean +/- SD 7.59 +/- 0.14) and maximal transport capacity of the antiport (Vmax 87.7 +/- 24.9 mmol.1-1.min-1) were higher in diabetic subjects with albuminuria compared to normotensive control subjects (pH 7.44 +/- 0.09; Vmax 55.6 +/- 10.3 mmol.l-1.min-1; p less than 0.001 for both), similar to the defect described in essential hypertension. These differences were not seen in diabetic subjects with normal urinary albumin/creatinine ratios (pH 7.46 +/- 0.09; Vmax 61.0 +/- 13.6 mmol.l-1.min-1). Buffering characteristics of the leucocytes at different pH in the Type 1 diabetic subjects with albuminuria differed from normal control subjects and diabetic subjects with normal urinary albumin/creatinine ratios. We conclude that increased leucocyte Na+/H+ antiport activity, a known marker of essential hypertension, is usually associated with nephropathy in Type 1 diabetes.

Glutamine transport by human intestinal basolateral membrane vesicle

This study characterizes for the first time, by use of a well-validated technique, glutamine transport across human basolateral membrane vesicles. Glutamine transport represented uptake into an osmotically active intravesicular space without significant metabolism. Glutamine uptake was temperature- and pH-dependent with maximal uptake at pH 7.5, and it was mediated by sodium-dependent and -independent processes. The initial rate of uptake was linear up to 20 s, as depicted by the formula gamma (nmol/mg protein) = 0.0009 X (s) - 0.0011 (r = 0.99). Kinetic analysis of glutamine uptake at concentrations between 0.01 and 0.3 mmol/L at 5 s under sodium and potassium gradients showed a maximal transport capacity (Vmax) of 0.39 +/- 0.04 and 0.21 +/- 0.02 nmol.mg protein-1.5 s-1 for sodium-dependent and -independent processes, respectively (p less than 0.01). Km values were 0.17 +/- 0.04 and 0.06 +/- 0.2 mmol/L, respectively (p less than 0.05). Glutamine uptake under the sodium-gradient condition was electrogenic whereas under the potassium-gradient it was electroneutral. Neutral amino acids inhibited both sodium-dependent and -independent processes. This study confirms and characterizes the presence of carrier-mediated glutamine uptake at the basolateral membranes of human enterocytes.

Lactate transport in isolated mouse muscles studied with a tracer technique—kinetics, stereospecificity, pH dependency and maximal capacity

Lactate transport across the sarcolemma of isolated mouse muscles was studied with a 14C tracer technique. The cellular tracer uptake could be inhibited by unlabelled L-lactate (and pyruvate) and to a lesser extent by D-lactase. The stereospecific fraction had a Km of 3.5 mM, and made up 50% of the total transport. The tracer uptake was unaffected by 0.05 mM DIDS and 0.2 mM amiloride, but was inhibited by cinnamate (Ki = 8 mM) and PCMBS (Ki = 0.8 mM). With high concentrations of the latter inhibitor compounds or with high concentrations of unlabelled L-lactate, the tracer uptake was inhibited 80%, which indicates that the main part of the transport involves facilitated diffusion. The remaining fraction (20%) was non-saturable, reduced at high pH, and could not be inhibited; it is probably mediated by diffusion of undissociated lactic acid. Lactate transport was pH-dependent, which is consistent with a lactate-H+ symport. The maximal transport capacity, as calculated from the pH changes measured with pH-sensitive micro-electrodes while the lactate gradient was 30 mM, was 11.8 mmol kg-1 min-1 (pH 6.2).

Pharmacokinetics and saturable renal tubular secretion of zidovudine in rats

The purpose of this study was to assess the effects of dose on the pharmacokinetics of zidovudine (3′-azido-3′-deoxythymidine; AZT) in rats. Zidovudine (AZT) was administered intravenously at doses of 10, 50, 100, and 250 mg/kg. Plasma and urine AZT concentrations were determined by HPLC. Plasma AZT concentrations declined rapidly with a terminal half-life ranging from 0.76h at a dose of 10 mg/kg to 1.58h at 250 mg/kg. Total clearance (CLT) was similar at the doses of 10 and 50 mg/kg, with values of 2.80 and 2.73 L/h/kg, respectively. However, there was a trend toward nonlinearity at the dose of 100 mg/kg (CLT = 2.13 L/h/kg) and a significant decrease in CLT (1.22 L/h/kg) at the dose of 250 mg/kg. Nonrenal clearance remained unaffected by dose with a mean value of 0.98 L/h/kg. Renal clearance (CLR) was similar at the doses of 10 and 50 mg/kg, with values of 1.89 and 1.37 L/h/kg, respectively. However, significant decreases in CLR were observed at the doses of 100 (CLR = 1.30 L/h/kg) and 250 mg/kg (CLR = 0.57 L/h/kg). The maximum transport capacity (Tmax) and the Michaelis-Menten constant (Km) for renal tubular secretion obtained after simultaneously fitting plasma concentration—time profiles at the four doses to a renal clearance model were 215.5 ± 82.1 mg/h and 119.3 ± 80.5 mg/L, respectively, thereby yielding an unbound secretory intrinsic clearance (CLus,int) of 1.81 L/h. The high Tmax and Km values account for the high CLR of AZT and explain the linearity of CLR over a wide range of AZT plasma concentrations. Active tubular secretion of AZT, however, is saturable at very high AZT concentrations, resulting in decreased CLR and, thus, CLT at higher doses. The steady-state volume of distribution (Vdss) was similar at the doses of 10 and 50 mg/kg (1.57 and 1.66 L/kg, respectively). There was a pattern towards decrease in Vdss at the dose of 100 mg/kg (Vdss = 1.17 L./kg) and a significant decrease at the dose of 250 mg/kg (Vdss = 0.81 L/kg) suggesting saturable tissue uptake or binding. Thus, the pharmacokinetics of AZT behaves linearly at low doses, but becomes dose dependent as the dose is further increased.

Vitamin D-regulated, ATP-dependent calcium transport by intestinal Golgi vesicles during maturation in the rat.

The developmental aspects of calcium uptake by intestinal Golgi vesicles was determined using highly purified Golgi vesicles from enterocytes of suckling (2 wk old), weanling (3 wk old), and adolescent (6 wk old) rats. Calcium uptake by Golgi vesicles at all age groups represented transport into the intravesicular space as evident by temperature dependency and by calcium ionophore A23187-induced calcium efflux studies. Calcium uptake was driven by ATP at all age groups, however, maximal uptake at 15 min was significantly greater in Golgi vesicles of adolescent rats compared to mean values in Golgi vesicles of suckling rats (p less than 0.01). Calcium uptake in the absence of ATP was minimal. The requirement for the adenine base and the hydrolysis of the beta-gamma-phosphodiester was tested by replacement of ATP in the incubation media by CTP and the nonhydrolyzable ATP analogue, adenylyl-(beta-gamma-methylendiphosphonate). Both agents had no stimulatory effect on calcium uptake. Calcium uptake was linear up to 40 s. Kinetic parameters of calcium uptake at free calcium concentrations of 0.04 to 1.0 microM showed a maximal transport capacity of 0.99 +/- 0.05, 0.55 +/- 0.04, and 0.29 +/- 0.03 nmol/mg protein/15 s for adolescent, weanling, and suckling rats, respectively. Km values were 0.16 +/- 0.02, 0.12 +/- 0.03, and 0.07 +/- 0.02 microM for adolescent, weanling and suckling rats, respectively. Km and Vmax values were significantly different between adolescent and suckling rats (p less than 0.01). The calcium regulatory protein calmodulin has no effect on calcium uptake by Golgi vesicles. Vitamin D deficiency in all age groups decreased ATP-dependent calcium uptake. Administration of 1,25-(OH)2 vitamin D3 8 h before death enhanced ATP-dependent calcium uptake in all age groups studied. This enhancement was the result of increase in maximal transport capacity of ATP-dependent calcium uptake. This study demonstrates a vitamin D-regulated ATP-driven calcium uptake by intestinal Golgi vesicles at all age groups including the suckling period. This transport system shows developmental patterns in regard to its kinetic parameters.