Microliters Min-1 Research Articles

Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye

Contractile properties of isolated trabecular meshwork strips have recently been described. In the present paper we characterize the regulation of the outflow pathway in the isolated perfused anterior segment of the bovine eye. Anterior segments of bovine eyes with detached iris, ciliary body and ciliary muscle were perfused at constant pressure of 8.8 mmHg. A constant outflow of approximately 6-8 microliters min-1 could be obtained for at least 3 hr. The calculated outflow resistance was in the range 1.1-1.4 mmHg min microliter-1. The relative outflow was significantly reduced after application of carbachol, reaching a maximal inhibition of 30%. EC50 for carbachol was 3 x 10(-8) mol l-1. Atropin completely blocked the effect of carbachol on outflow. Morphological examination of perfused anterior segments which were perfused with carbachol revealed an intact fine structure of the meshwork cells. Pilocarpine at 10(-5) mol l-1 reduced outflow by 15%. Epinephrine at 10(-5) mol l-1 reduced outflow, while epinephrine at 10(-6) mol l-1 slightly increased the outflow rate. This effect could be blocked by metipranolol. Endothelin-1 in concentrations of 2 x 10(-9) and 2 x 10(-8) mol l-1 inhibited relative outflow by > 30%. Carbachol, pilocarpine, endothelin and a high dose of epinephrine, which have been shown to induce contractions in isolated bovine trabecular meshwork and ciliary muscle strips, induced a reduction of outflow rate and an increase of outflow resistance of the anterior segment. Thus, at least in the bovine eye, the trabecular meshwork per se is directly involved in the regulation of aqueous humor outflow.

Dentinal fluid dynamics in human teeth, in vivo

Cavities were prepared in human premolars scheduled for extraction for orthodontic reasons. The smear layer was removed from the dentin surface by acid etching, and the cavity was sealed using a hollow chamber. The chamber was filled with sterile saline solution and connected via tubing to a hydraulic circuit featuring an adjustable pressure reservoir and a device that measures fluid movement across dentin. In the absence of any exogenous pressure, all cavities exhibited an outward fluid flow rate of 0.36 microliters min-1 cm-2. As exogenous pressure was applied to the cavity, the outward flow slowed. The exogenous pressure that stopped outward fluid flow was taken to be equal to normal pulpal tissue pressure. The mean value was 14.1 cm H2O in five teeth. This simple method permits measurement of dentinal fluid flux, the hydraulic conductance of dentin, and estimates pulpal tissue pressure.

Differentiation- and Protein Kinase C-dependent Regulation of Alanine Transport via System B

The regulation of sodium-dependent L-alanine transport is described for the first time in intestinal cells. Substrate analogue inhibition patterns and Dixon analyses indicated that uptake occurred via transport system B, an epithelial cell variant of systems B0,+ and ASC. System B served > 95% of the Na(+)-dependent alanine uptake in both undifferentiated (2 days postpassaging) and differentiated (> 9 days postpassaging) states of the human Caco-2 cultured intestinal cell line. (Methylamino)isobutyric acid-inhibitable system A transport accounted for < 5% of total alanine uptake. System B activity was greater in undifferentiated cells compared with the differentiated state, and activity at any differentiation state was stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA). The maximal stimulation, determined by TPA dose-response/exposure time data, was attributable to a change in cell transport capacity (Vmax), with Km unaffected. The Vmax of system B was greater in 2-day-old cells (2.79 +/- 0.21 nmol min-1 mg of protein-1; Km = 164 +/- 26 microM alanine), decreasing to Vmax = 0.51 +/- 0.03 nmol min-1 mg of protein-1 (Km = 159 +/- 14 microM) in 9-day-old cells. Regardless of differentiation status, the sodium-activation Hill coefficient was 1.06 +/- 0.10, and the alanine passive diffusion permeability coefficient was 0.53 +/- 0.08 microliter min-1 mg of protein-1. Phorbol ester up-regulated the Vmax of system B in 2-day-old cells to Vmax = 6.32 +/- 0.37 nmol min-1 mg of protein-1 (Km = 169 +/- 18 microM), and in 9-day-old cells to Vmax = 1.42 +/- 0.05 nmole min-1 mg of protein-1 (Km = 180 +/- 10 microM). Phorbol ester stimulation of transport occurred after at least 6 h of continual exposure, and was blocked by the protein kinase C inhibitors chelerythrine or photoactivated calphostin C. Protein synthesis inhibitors cycloheximide and actinomycin D each blocked the phorbol ester up-regulation of system B activity. It is concluded that Caco-2 cells regulate carrier-mediated sodium-dependent transport of L-alanine by changing the membrane capacity to transport alanine via system B and that this regulation involves de novo protein synthesis under the control of protein kinase C.

Chloride-activated water permeability in the frog corneal epithelium.

We have previously reported that the isolated frog corneal epithelium (a Cl(-)-secreting epithelium) has a large diffusional water permeability (Pdw approximately 1.8 x 10(-4) cm/s). We now report that the presence of Cl- in the apical-side bathing solution increases the diffusional water flux, Jdw (in both directions) by 63% from 11.3 to 18.4 microliters min-1.cm-2 with 60 mM [Cl] exerting the maximum effect. The presence of Cl- in the basolateral-side bathing solution had no effect on the water flux. In Cl(-)-free solutions amphotericin B increased Jdw by 29% but only by 3% in Cl(-)-rich apical-side bathing solution, suggesting that in Cl(-)-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br- (75 mM) also increased Jdw by 68%. The effect of Cl- on Jdw was observed within 1 min after its addition to the apical-side bathing solution. HgCl2 (0.5 mM) reduced the Cl(-)-increased Pdw by 31%. The osmotic permeability (Pf) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10(-3) cm/s) in Cl(-)-free and Cl(-)-rich apical-side bathing solutions respectively. It seems that apical Cl-, or Cl- secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl-, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.

Determination of substrates using poly(ethylene glycol)-stabilized dehydrogenase enzymes by microlitre per minute flow injection.

Flow injection (FI), at a flow rate of microliter min-1, is an effective method for enzymic substrate determination using low concentrations of poly(ethylene glycol) (PEG)-stabilized soluble enzymes. PEG stabilizes dehydrogenase enzymes for at least several days by promoting sub-unit association. Band broadening of knitted open tubular reactors is reduced as flow rate decreases below 300 microliter min-1 and a small tubing diameter is important for a faster rate of absorbance signal increase with residence time. Small (0.5 microliter) sample injections also ensure narrow FI peaks. The determination of several substrates such as pyruvate, lactate, and cortisone using appropriate PEG-stabilized enzymes is demonstrated with this FI instrument at 25 or 50 microliters min-1 with sample throughputs of the order of 2-3 min per sample. The determination of lactate in serum samples is also possible. The advantage of this method, sample throughput, is not sacrificed but enzyme consumption is considerably less, compared to standard ml min-1 FI.

Determination of the pseudofacility by fluorophotometry in the human eye.

Pseudofacility (decrease in aqueous humor flow in response to increase in the intraocular pressure (IOP)) has been evaluated in animals and in humans by a combination of different techniques that seriously alter the ocular physiology, such as the cannulation of the eyeballs, use of radioactive isotopes, perilimbic suction cups or tonography. Very different results have been obtained. Our objective is to relate the value of the aqueous humor flow in humans (as measured by fluorometry) with IOP. In order to obtain the widest range of IOP possible, we have performed this study in both normal as well as glaucoma patients. 48 eyes were studied, corresponding to 31 subjects (mean age 58 +/- 14 years). Of these, 21 corresponded to normal subjects and 27 to glaucoma patients. The aqueous humor flow (F) was determined by fluorophotometry (Protocol of Yablonsky) and the IOP was measured thereafter. The mean value of F was 2.1 +/- 0.5 microliter/min for healthy eyes and 2.2 +/- 0.7 microliter/min for glaucoma-affected eyes. The values were not different (p > 0.1). The mean value of the IOP was 15.3 +/- 3.1 mmHg in the normal population and 22.1 +/- 4.3 mmHg in the glaucoma patients. A linear relationship between F and IOP was found (r = -0.41, p < 0.05, decrease in F = 0.081 microliter min-1 mmHg-1). The value of the pseudofacility in the humans can be estimated at 0.081 microliter min-1, mmHg-1, which constitutes 27% of the total outflow facility. This figure also makes it possible to estimate the role of the ultrafiltration in blood in the production of the aqueous humor since it is assumed that active secretion is pressure-independent. The results obtained are concordant with a secondary but no unimportant role of the plasmatic ultrafiltration with respect to the active secretion.

Somatosensory regulation of renal function in the stroke‐prone spontaneously hypertensive rat.

1. Chloralose-urethane-anaesthetized Wistar rats and stroke-prone spontaneously hypertensive rats (SHRSP) were prepared for measuring renal function. 2. Bilateral brachial nerve stimulation for 15 min, at 1.3 Hz (15 V, 0.2 ms), increased blood pressure from 119 +/- 2 to 151 +/- 6 mmHg (P < 0.01) in Wistar rats and from 181 +/- 6 to 203 +/- 6 mmHg (P < 0.05) in SHRSP; renal perfusion pressure was mechanically regulated at prestimulus levels but renal blood flow (4.1 +/- 0.7 ml min-1 g-1 in Wistar rats and 3.4 +/- 0.3 ml min-1 g-1 in SHRSP) and glomerular filtration rate (1.18 +/- 0.09 ml min-1 g-1 in Wistar rats and 0.88 +/- 0.01 ml min-1 g-1 in SHRSP) decreased between 5 and 30% (P < 0.05-0.001). 3. During brachial nerve stimulation, urine flow (13.8 +/- 2.2 microliters min-1 g-1) and absolute and fractional sodium excretion (3.70 +/- 0.64 mumol min-1 g-1 and 2.06 +/- 0.23%) decreased by 60-70% in the Wistar rats (P < 0.01-0.001) and the responses were unaffected by vagotomy or carotid sinus denervation. In the SHRSP, urine flow rate (9.74 +/- 2.52 microliters min-1 g-1) and absolute and fractional sodium excretion (1.98 +/- 0.59 mumol min-1 g-1 and 1.41 +/- 0.28%) decreased by 15-30% (P < 0.05-0.01), decreases which were significantly (P < 0.05) smaller than those observed in the Wistar rats. Moreover, the responses were some 40-60% larger in the vagotomized and carotid sinus-denervated SHRSP. 4. The influence of vagal and carotid sinus afferent nerves on the somatosensory-induced renal responses in the Wistar rats was small, whereas in the SHRSP they appeared to exert a marked tonic inhibitory effect. It is concluded that there is excessive baroreceptor restraint on neural regulation of renal function in the SHRSP.

Determination of xanomeline in human plasma by ionspray tandem mass spectrometry

Xanomeline is a muscarinic receptor agonist currently in phase II clinical trials for the treatment of Alzheimer's disease. A fast, sensitive and specific assay has been developed to determine xanomeline plasma concentrations using ion-spray tandem mass spectrometry. Xanomeline and a structural analog, LY282122, were extracted from basifed plasma into hexane. The dried hexane extracts were reconstituted and injected onto a 10 x 1 mm C18 reversed-phase column. A mobile phase of 33 mM ammonium acetate and 0.33% acetic acid in 30/70 (v/v) water-acetonitrile was pumped through the column at 50 microliters min-1. The mobile phase eluant was introduced directly into the ion-spray interface. The mass spectrometer was operated in the positive ion mode for specific detection of the product ions of xanomeline and the internal standard. The method has a linear range of 0.075-5.0 ng xanomeline per milliliter of plasma. Sample run times were 2.5 min from one injection to the next.

Effects of a nitric oxide donor on glutamate and GABA release in striatum and hippocampus of the conscious rat.

The effects of intracerebral perfusion of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) and the nitric oxide synthase inhibitor N-nitroarginine (NARG) on the extracellular concentrations of glutamate (GLU) and gamma-aminobutyric acid (GABA) in striatum and CA1 area of the hippocampus were studied. Continuous push-pull perfusions at a flow rate of 20 microliters min-1 were performed in the conscious rat. SIN-1 (100, 200, and 400 microM) and NARG (100 microM) were perfused over 20 min. In both striatum and CA1 SIN-1 increased extracellular concentrations of GLU (maximal increase 150% and 197% of baseline, respectively) and GABA (maximal increase 202% and 204% of baseline, respectively). NARG had no effects on extracellular levels of GLU and GABA in either area. These results are consistent with the hypothesis that NO acts as a modulator of GLU and GABA release in both striatum and hippocampus. This study is the first to report the potentiation of GLU and GABA release by NO in CA1 area of the hippocampus in the conscious rat.

A comparison between endogenous and exogenous lithium clearance in the anaesthetized rat

The present study tests whether the endogenous renal lithium clearance (CLi) may be used as an alternative to the conventional method in order to avoid the adverse effects of exogenous lithium on renal function. Endogenous CLi) was measured by flameless atomic absorption spectrometry (AAS) in halothane anaesthetized, laparotomized rats and compared with values obtained after subsequent intravenous (i.v.) administration of LiCl. Endogenous plasma lithium concentration (PLi) was 0.30 +/- 0.06 mumol l-1. When measured by both conventional flame emission AAS and flameless AAS on the same samples, values for PLi did not differ significantly. Endogenous CL1/GFR was 0.195 +/- 0.01 (mean +/- SEM). Amiloride caused on significant change in either the endogenous CLi or 51Cr-EDTA clearance, a measure of glomerular filtration rate (GFR), but it caused a significant increase in CNa. Administration of exogenous Li+ had no significant effect on CLi (230 +/- 33 vs. 234 +/- 35 microliters min-1 gKW-1); both urine flow and CNa increased significantly (P < 0.01) (5.4 +/- 0.4 vs. 9.6 +/- 1.3, and 2.4 +/- 0.5 vs. 10.7 +/- 2.4 microliters min-1 gKW-1, respectively). GFR and calculated proximal tubular reabsorption rate (APR) both decreased by approximately 100 microliters min-1 gKW-1 (both P < or = 0.025), suggesting a moderate inhibition of APR after the acute elevation of plasma Li+ concentration. It is concluded that the endogenous CLi may be used as an alternative to the conventional method.

Glucose Polymer in the Fluid Therapy of Acute Diarrhoea: Studies in a Model of Rotavirus Infection in Neonatal Rats

1. Unlike standard glucose-electrolyte oral rehydration solutions, solutions containing polymeric glucose as substrate can significantly reduce stool output, duration of diarrhoea and total oral rehydration solution requirements. However, neither the underlying mechanisms nor the optimal size and concentration of glucose polymer has been defined. 2. We have used a model of rotavirus diarrhoea in neonatal rats to compare the effects on water and solute absorption of varying the concentration of a glucose polymer (mean chain length five glucose residues) in experimental oral rehydration solutions. Three polymer (P) solutions were compared with solutions of identical electrolyte content (mmol/l: sodium, 60; potassium, 20; chloride, 60; citrate, 10) containing equivalent amounts of free glucose (G) as substrate by perfusion of the entire small intestine in situ. The polymer (9, 18, 36 mmol/l; 159, 168, 186 mosmol/kg, respectively) and the monomer (45, 90, 180 mmol/l; 195, 240 320 mosmol/kg) solutions were perfused in normal and rotavirus-infected neonatal rats. 3. In normal intestine polymer solutions promoted greater water absorption [P9, mean 291.4 (SEM 16.4); P18, 331.9 (13.1); P36, 284.3 (11.8) microliters min-1 g-1] than their equivalent monomer solutions [G45, 220.8 (8.4); G90, 240 (21); G180,79.4 (14.5) microliters min-1 g-1; P < 0.02]. In rotavirus-infected intestine, water absorption from all solutions declined, but the fall was much less pronounced from the polymer solutions [P9, 232.8 (6); P18, 277.2 (20.5); P36, 166 (18.2) microliters min-1 G-1] than from their monomeric counterparts [G45, 116.7 (25.5); G90, 68.7 (12.4); G180, 21 (11.6) microliters min-1 g-1; P < 0.005].(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of chronic sodium depletion on renal function in conscious rats.

In order to investigate the effect of chronic sodium depletion on renal proximal tubular reabsorption, studies were performed in conscious, unrestrained Brattleboro rats. Since these animals lack circulating vasopressin, fractional water reabsorption in the distal nephron can be assumed to be constant and changes in urine flow rate should therefore reflect changes in end-proximal fluid delivery. Sodium depletion was induced by placing rats on a low-sodium diet (4 mmol Na (kg dry wt)-1) and administering frusemide (40 mg (kg body wt)-1) by gavage on the first 2 days. Extracellular volume, measured after 7-9 days, was reduced by 19% (P < 0.02) as compared with that of rats maintained on a control diet. Urine flow rate, measured during days 4-7 of the low-sodium diet, was significantly lower than that of control rats (142 +/- 8 vs. 168 +/- 5 ml day-1, P < 0.01). Since renal papillary interstitial fluid osmolality was found to be reduced in the sodium-depleted rats (693 +/- 38 vs. 812 +/- 36 mosmol (kg H2O)-1, P < 0.05), it is unlikely that water reabsorption from sites beyond the proximal tubule had increased. The observed reduction in urine flow rate therefore strongly suggests a reduction in end-proximal fluid delivery. In the second part of the study, a single group of Brattleboro rats was used, in which osmotic minipumps were implanted in the peritoneal cavity for continuous infusion of [14C]inulin. After recovery from the operation, the rats were maintained on a control diet for 6 days (pre-control period), then subjected to sodium depletion (low-sodium diet for 6 days, frusemide administration on the first 2 days), and finally returned to the control diet for 6 days, with access to 0.46 M NaCl solution on the first 2 days, in order to restore sodium balance (post-control period). On the final 2 days of each phase, urine flow rate and [14C]inulin clearance (= glomerular filtration rate, GFR) were measured. Urine flow rates during the pre-control, sodium depletion and post-control periods were 169 +/- 7, 132 +/- 8 (P < 0.001) and 176 +/- 8 microliters min-1, respectively; corresponding values for fractional water excretion were 7.0 +/- 0.3, 6.0 +/- 0.5 (P < 0.01) and 7.4 +/- 0.4%. Only a small reduction GFR, of borderline statistical significance, was observed during sodium depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrarenal haemodynamic and glomerular responses to inhibition of nitric oxide formation in rabbits.

1. The renal effects of inhibiting nitric oxide (NO) formation using N-nitro-L-arginine (NOLA, 20 mg kg-1) were examined using micropuncture techniques in pentobarbitone-anaesthetized rabbits. 2. Renal vascular resistance doubled from 2.7 +/- 0.5 to 5.0 +/- 1.1 mmHg ml-1 min-1 after NOLA (P < 0.01), with similar percentage increases in both pre- (149 +/- 38%, P < 0.01) and postglomerular (158 +/- 42%, P < 0.01) resistance. 3. Glomerular capillary pressure rose from 33 +/- 1 to 40 +/- 1 mmHg after NOLA (P < 0.01) but despite this, glomerular filtration rate (GFR) and single nephron glomerular filtration rate did not significantly change. 4. Blood pressure increased 18 +/- 1 mmHg (P < 0.001) within 10 min of NOLA administration and remained near this level for the next 90 min. 5. The glomerular ultrafiltration coefficient (Kf) decreased significantly from 0.085 +/- 0.022 to 0.035 +/- 0.006 nl s-1 mmHg-1 (P < 0.05). 6. Urine flow and sodium excretion increased markedly (26 +/- 9 to 337 +/- 102 microliters min-1 and 5 +/- 2 to 342 +/- 12 mumol min-1 respectively, (P < 0.001)) and sodium fractional excretion rose from 1.0 +/- 0.3 to 8.0 +/- 2.2% (P < 0.01). 7. Thus, administration of NOLA to rabbits caused vasoconstriction of both pre- and postglomerular vessels, diuresis and natriuresis without significant change in GFR, and a reduction in Kf. The results suggest that NO may play an important role in the regulation of renal haemodynamics and glomerular function.

Interstitial glucose and lactate balance in human skeletal muscle and adipose tissue studied by microdialysis.

1. Microdialysis was used to gain insight into the substrate exchanges in the interstitial space of skeletal muscle and adipose tissue. Probes were inserted in the quadriceps femoris muscle and para-umbilical subcutaneous adipose tissue of thirteen subjects and microdialysis was performed at different flow rates (1-4 microliters min-1) and during changes in tissue blood flow. 2. When ethanol (5 mM) is included in the perfusion solution, the ethanol clearance from the probe is a measure of tissue blood flow. Blood flow changes induced by adenosine or vasopressin perfusion, by exercise or by circulatory occlusion resulted in ethanol clearance values of 69-139% of the basal level. The ethanol clearance was higher in skeletal muscle than in adipose tissue (32-62%, P < 0.001), a difference compatible with a higher blood flow in muscle tissue. 3. The fraction of the interstitial glucose concentration that was recovered with the microdialysis was similar in skeletal muscle (the absolute values being 1.70 +/- 0.14 mM at 1 microliter min-1 and 0.59 +/- 0.05 mM at 4 microliters min-1) and adipose tissue (1.89 +/- 0.20 mM at 1 microliter min-1; 0.54 +/- 0.05 mM at 4 microliters min-1) and correlated inversely with the tissue ethanol clearance, both in the basal state and during changes in tissue blood flow (muscle: r = -0.56 to -0.67; adipose tissue r = -0.72 to -0.95). Coefficients of variation were 6-8% (glucose) and 11-16% (lactate) and were similar during isometric exercise. The reproducibility of the technique (comparison of two contralateral probes; perfusion flow rate 4 microliters min-1) was 5.3-8.3% (ethanol) and 23.9-20.8% (glucose) in muscle (n = 6) and adipose tissue (n = 4) respectively. 4. The skeletal muscle dialysate lactate concentration (1 microliter min-1: 1.16 +/- 0.2 mM) was higher than in adipose tissue (0.76 +/- 0.08 mM, P < 0.05), where the absolute amount of lactate that could be removed from the tissue (at 4 microliters min-1) was only half of that in skeletal muscle (0.8 +/- 0.11 vs. 1.76 +/- 0.23 nmol min-1, P < 0.05). The dialysate lactate level was not affected in either tissue by large changes in the interstitial glucose concentration indicating that in neither tissue is blood glucose a significant source of lactate formation. 5. The blood flow effects on the dialysate glucose concentration are the likely consequence of probe glucose drainage artificially shifting the balance between the supply and consumption of interstitial glucose.(ABSTRACT TRUNCATED AT 400 WORDS)

Osmotic barrier properties of the rat peritoneal membrane.

In this study the osmotic barrier characteristics of the rat peritoneal membrane were investigated. Fluid movements between the peritoneal cavity and the blood were measured following instillation of isotonic saline (control) and hypertonic solutions of NaCl, glucose, sucrose, raffinose and myoglobin (test solutions). Moreover, 5 and 8% albumin in NaCl were investigated. Osmotic transients were assessed using a simple volume recovery technique. Peritoneal osmotic conductances (i.e. products of peritoneal hydraulic conductances [LpS] and solute reflection coefficients [sigma]) were calculated from the differences in the rates of peritoneal fluid loss and in osmotic pressures between test solutions and the isotonic saline control solution. The osmotic conductance to glucose was estimated to be 1.63 microliters min-1 mmHg-1 m-2 and that for albumin to be 59.6 microliters min-1 mmHg-1 m-2. Assuming an albumin sigma of 0.9, the sigma of glucose was estimated to be 0.025, in accordance with previous measurements for the cat peritoneal membrane. The osmotic conductances assessed here were compatible with an 'overall' peritoneal equivalent small pore radius of 47-48 A, but could also be fitted to a three-pore model of peritoneal permselectivity, including a transcellular (ultra-small pore) pathway and a large pore pathway. The great discrepancy between peritoneal sigma for small solutes and that for albumin obtained in this study indicates that small solute reflection coefficients are close to zero while that for albumin is not far from unity. Furthermore, the peritoneal hydraulic conductance (ultrafiltration coefficient) is large enough to allow for a substantial absorption of fluid directly into the plasma when the crystalloid osmotic pressures in blood and peritoneal dialysate are in equilibrium.

Evidence for active maternal-fetal transport of Na+ across the placenta of the anaesthetized rat.

1. In order to investigate mechanisms of Na+ transfer, the unidirectional maternal-fetal clearance (Kmf) of 22Na+ and of 51Cr-EDTA (a marker of paracellular diffusion) was measured across the intact or umbilically or dually perfused placenta of the anaesthetized rat. 2. The Kmf of 22Na+ in the intact preparation (18.5 +/- 2.7 microliters min-1, mean +/- S.D., n = 105 placentas) exceeded that of 51Cr-EDTA in the same experiments (1.4 +/- 0.3 microliters min-1) by more than ten times, whereas the difference in their diffusion coefficients in water was only 2-fold. In the perfused preparations the difference in the Kmf values was 6-fold. 3. Assuming that a simple model of paracellular diffusion through wide pores was one component of transfer, the Kmf of 51Cr-EDTA and the diffusion coefficients were used to calculate a component of 22Na+ clearance (Kmf,residual) and of Na+ flux (Jmf,residual) across the perfused placentas which could not be accounted for by transfer through the paracellular route. 4. Kmf,residual of 22Na+ across the dually perfused placenta was significantly lower when temperature was reduced, the temperature quotient (Q10) of the transfer being about 2. Kmf,residual was also significantly lower when 0.1 mM ouabain was perfused on the fetal side. Jmf,residual exhibited saturation kinetics characterized by an apparent Michaelis constant (Km) of 90 mM. Kmf,residual was not influenced by 0.5 mM frusemide, 0.5 mM amiloride or by 0.5 mM hydrochlorothiazide administered to the maternal side. It was significantly increased by 1 mM alanine on the maternal side suggesting that the coupled transfer of Na+ and amino acids may contribute significantly to the maternal-fetal flux of Na+. 5. These observations suggest that most (80%) of the maternal-fetal flux of Na+ across the rat placenta is effected by active transcellular transport. This transport involves passive entry of Na+ into the trophoblast from the maternal side by a largely unknown saturable mechanism and active extrusion of Na+ from trophoblast to the fetal side by Na(+)-K(+)-ATPase.

Transcellular fluid secretion induced by cholera toxin and vasoactive intestinal polypeptide in the small intestine of the rat.

The permeation of intravenously administered 51Cr-EDTA and [14C]mannitol to the perfused intestinal lumen was measured in anaesthetized rats together with the net intestinal fluid. Net fluid secretion was induced by cholera toxin or by intravenous infusion of vasoactive intestinal polypeptide (VIP). The plasma clearance of Cr-EDTA and mannitol was 0.9 +/- 0.1 and 1.4 +/- 0.2 microliters min-1 g-1 intestine during the control period prior to the secretion and the net fluid absorption was about 7 +/- 5 microliters min-1 g-1. Cholera toxin induced a net fluid secretion of about 30 +/- 7 microliters min-1 g-1 but the clearance did not rise but decreased significantly. The findings for VIP-induced secretion were similar. No indication of solvent drag was seen. Thus it is concluded that the fluid was secreted in channels which were smaller than the probes and we propose that the secreted fluid entered the intestinal lumen through the epithelial cells and not by the paracellular route. The decreased permeation of Cr-EDTA and mannitol from plasma to lumen during volume secretion suggest that there was a decreased mucosal permeability during the secretion. The decrease in permeability was consistent with a decrease in pore size. One explanation of the data is that the pore radius contracted from about 35 to 15 A during cholera if we assume a homogenous pore population. However, the data indicated that there was not a uniform size of the pore.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetic studies in mice with ICI D0490, a novel recombinant ricin A-chain immunotoxin

A colorectal tumour-directed immunotoxin, ICI D0490, has been constructed by linking recombinant ricin A-chain to C242, a mouse monoclonal antibody, by means of a methyl-hindered disulphide bond. Recombinant ricin A-chain and a hindered disulphide linker were anticipated to confer favourable pharmacokinetic properties on the immunotoxin. The pharmacokinetics of ICI D0490 have been studied in mice following single and repeated i.v. administration. The concentrations of intact immunotoxin in mouse plasma at various time intervals after injection for up to 96 h were measured by a solid-phase enzyme-linked immunosorbent assay (ELISA) and the data analysed by both model-dependent (two compartment) and model-independent methods. Following a single i.v. bolus dose of 2.5 mg kg-1 (50% of the LD10 in mice), the clearance of ICI D0490 from the plasma was extremely slow; 34 microliters min-1 kg-1, t1/2 beta = 33 h. Model-dependent and model-independent analyses gave comparable results with steady state volumes of distribution of 93 and 69 ml kg-1, respectively. The two compartment analysis gave an initial volume of distribution (63 ml kg-1) which is consistent with the predicted plasma volume. Over the dose range 0.05-5 mg ICI D0490 kg-1, plasma levels at 2 and 24 h were linearly related to dose (r > or = 0.98) indicating that at doses up to 5 mg ICI D0490 kg-1 clearance does not appear to have a saturable component. Repeated doses of ICI D0490 (1 mg kg-1 day x 5) did not lead to drug accumulation. These studies demonstrate that ICI D0490 has excellent in vivo stability and persistence which, in conjunction with activity and toxicity data, identify ICI D0490 as a promising candidate for clinical evaluation in the treatment of colorectal cancer.

Lymphocyte subsets recirculate from blood to lymph at different rates in conscious sheep

Lymphocytes migrate and recirculate continuously between blood and lymph. The current study compared the migratory properties of lymphocyte subsets by labelling lymphocyte surface antigens and analysing simultaneous samples of mesenteric lymph, systemic arterial blood and mesenteric venous blood from conscious sheep. The lymphocyte output in lymph averaged 5.33 +/- 0.89 x 10(6) min-1 (lymph flow rate of 92 +/- 13 microliters min-1, lymphocyte count 61.9 +/- 10.7 x 10(9) l-1, n = 10) and the proportion of cells which carried the CD4 antigen (CD4+) was considerably higher in lymph (46 +/- 3%) than in arterial blood (23 +/- 2%; p < 0.01, n = 9). This increase was paralleled by a small but statistically significant decrease in the percentage of CD4+ cells in mesenteric venous blood (20 +/- 2%), as compared with systemic arterial blood (26 +/- 2%; p < 0.05, n = 6), which is consistent with the hypothesis that CD4+ cells migrate out of the blood vessels, and recirculate through the lymphatics, more readily than other lymphocyte subsets.

The ethanol technique of monitoring local blood flow changes in rat skeletal muscle: implications for microdialysis.

We have investigated the feasibility of monitoring local skeletal muscle blood flow in the rat by including ethanol in the perfusion medium passing through a microdialysis probe placed in muscle tissue. Ethanol at 5, 55, or 1100 mM did not directly influence local muscle metabolism, as measured by dialysate glucose, lactate, and glycerol concentrations. The clearance of ethanol from the perfusion medium can be described by the outflow/inflow ratio ([ethanol]collected dialysate/[ethanol]infused perfusion medium), which was found to be similar (between 0.36 and 0.38) at all ethanol perfusion concentrations studied. With probes inserted in a flow-chamber, this ratio changed in a flow-dependent way in the external flow range of 5-20 microliters min-1. The ethanol outflow/inflow ratio in vivo was significantly (P less than 0.001) increased (to a maximum of 127 +/- 2.8% and 144 +/- 7.4% of the baseline, mean +/- SEM) when blood flow was reduced by either leg constriction or local vasopressin administration, and significantly (P less than 0.001) reduced (to 62 +/- 6.4% and 43 +/- 4.4% of baseline) with increases in blood flow during external heating or local 2-chloroadenosine administration, respectively. Dialysate glucose concentrations correlated negatively with the ethanol outflow/inflow ratio (P less than 0.01) and consequently decreased (to 46 +/- 7.6% and 56 +/- 5.6% of baseline) with constriction and vasopressin administration and increased (to 169 +/- 32.5% and 262 +/- 16.7% of baseline) following heating and 2-chloroadenosine administration. Dialysate lactate concentrations were significantly increased (approximately 2-fold, P less than 0.001) during all perturbations of blood flow. In conclusion, this technique makes it possible to monitor changes in skeletal muscle blood flow; however, methods of quantification remain to be established. The fact that blood flow changes were found to significantly affect interstitial glucose and lactate concentrations as revealed by microdialysis indicates that this information is critical in microdialysis experiments.