Distribution pharmacokinetics of warfarin in the rat, a non-linear multicompartment model

Preliminary analysis and linear two-compartment solutions of warfarin plasma concentrations recorded in the rat after intravenous bolus injections of 1, 2, 8 and 40 mg/kg of sodium warfarin revealed marked non-linearities. The half-life of total warfarin concentration in the plasma from 1-12h remained unchanged with all the doses used, but that of free warfarin was shorter with 40 mg/kg, possibly as the result of an increase in the binding of the drug to plasma proteins as the high total warfarin concentration decreased. The apparent volume of distribution generally increased with increasing dose, and differed according to the method used for its calculation. Liver warfarin data could be solved with Langmuir type saturation kinetics, but the saturation phenomena were slight in the concentration range studied. A non-linear multicompartment model was constructed, the physiological spaces of which were plasma, interstitial fluid and tissue. The binding of free warfarin to plasma proteins, interstitial fluid proteins and tissue structures was assumed to occur instantaneously, with saturable binding to plasma and interstitial fluid proteins, and a constant binding to tissues. The fluxes between the free warfarin pools of plasma and interstitial fluid as well as elimination were assumed to be linear. Following parameters were simulated simultaneously, using an analog hybrid computer: two for the above-mentioned fluxes, four for zero time drug mass distribution between plasma and interstitial fluid, and one for tissue binding. According to the best fits, warfarin is preferentially distributed into plasma, interstitial fluid and highly perfused tissues. The solution suggests that non-linearities in the pharmacokinetics of warfarin, a highly plasma protein-bound drug, first occur in plasma and interstitial fluid. Therefore, it is believed that the quantitative non-linear multicompartment approach presented in this paper might be useful in studying the kinetic behaviour of other highly plasma protein-bound drugs, too.     

Considerations in the determination by microdialysis of resting extracellular amino acid concentrations and release upon spinal cord injury

The following issues are further addressed: (1) Is there considerable leakage of amino acids from the circulation into the space around microdialysis probes, or are amino acid concentrations naturally much higher in the interstitial space than is generally thought? (2) Do observed high interstitial concentrations or depletion of substances in the intracellular space by microdialysis affect release measurements upon spinal cord injury? Amino acid concentrations around microdialysis fibres in the spinal cord of rats were found to approach those in the circulation and to be much higher than interstitial concentrations previously estimated in the CNS. However, much lower concentrations of amino acids were derived in the hippocampus by analogous experiments. Considerable Evans Blue/albumin leaked from the circulation into the interstitial space in the spinal cord immediately after fibre insertion. However, this movement diminished considerably by 4 h later, demonstrating substantial resealing of the blood-brain barrier, at least to large molecules. There is either substantial damage-induced movement of amino acids from the circulation into the dialysis zone after insertion of a microdialysis probe, or there is much less impediment to movement of amino acids across the blood-brain barrier in the spinal cord than in the brain. At low flow rates through the fibre, adding concentrations of amino acids to the inside of the fibre equal to the concentrations around the fibre to prevent their depletion by removal through the microdialysis fibre did not affect increases in concentrations of amino acids in microdialysates following injury. Thus the high concentrations of amino acids present around microdialysis fibres following their insertion do not seem to disturb measurements of amino acid release upon spinal cord injury.     

Comparison of three different experimental methods for the assessment of peripheral compartment pharmacokinetics in humans

In many cases the concentration reached in a peripheral effect compartment rather than in plasma determines the clinical outcome of therapy. Therefore, several experimental approaches have been developed for direct assessment of drug kinetics in peripheral compartments. Particularly saliva sampling, skin blister fluid sampling, and in vivo microdialysis are frequently employed for measuring peripheral drug concentrations. However, data derived from these techniques have never been directly compared. In the present study, the tissue kinetics of theophylline were measured following single dose administration simultaneously in cantharides induced skin blisters, saliva and microdialysates of subcutaneous- and skeletal muscle- tissue and compared to plasma concentrations. Theophylline was administered to 9 healthy volunteers as an i.v. infusion of 240 mg. Mean ratio (AUCsaliva/AUCplasma) was 0.63 +/- 0.05, mean ratio (AUCblister/AUCplasma) was 0.69 +/- 0.12, mean ratio (AUCmuscle/AUCplasma) was 0.41 +/- 0.10, mean ratio (AUCsubcutaneous/AUCplasma) was 0.34 +/- 0.07. The time course of the concentration(peripheral)/concentration(plasma)-ratios showed that tissue concentrations obtained by microdialysis were closely correlated to free plasma levels, whereas saliva- and cantharides blister data overestimated the corresponding free plasma concentrations. It is concluded that microdialysis represents a reliable technique for the measurement of unbound peripheral compartment concentrations and is superior to saliva- and skin blister concentration measurements.     

Interstitial methotrexate kinetics in primary breast cancer lesions

The transfer of cytotoxic agents across the tumor endothelium into the interstitial tumor space is considered a critical step in clinical resistance of solid tumors to antineoplastic chemotherapy. However, experimental data on drug transfer from the blood into the interstitium of solid tumors are scarce. Therefore, in this study, we used an innovative technique, in vivo microdialysis, for measuring interstitial tumor pharmacokinetics and plasma-to-tumor transfer rates of methotrexate (MTX) in breast cancer patients. Microdialysis probes were inserted into the primary tumor and the periumbilical s.c. adipose layer of nine previously chemotherapy-naive breast cancer patients to monitor interstitial concentrations following i.v. administration of MTX (40 mg/m2) during a three-drug treatment regimen. Mean interstitial MTX load in breast tumors, expressed as area under curve (AUC), was 60 +/- 20% (mean +/- SE; coefficient of variation = 100%) of mean plasma MTX load. There was no correlation between plasma AUC and the AUC in the interstitial space of tumor tissue (P = 0.93). Not one of the parameters plasma, interstitial tumor load, and transfer rate of MTX to the interstitial space was associated with favorable clinical response. In conclusion, plasma levels of MTX are not predictive of intratumor levels. There is a high interindividual variability in transendothelial MTX transfer. Under the present conditions, access of MTX to the interstitial space is not a rate-limiting step for clinical response to chemotherapy.     

Interstitial fluid concentrations of glycerol, glucose, and amino acids in human quadricep muscle and adipose tissue. Evidence for significant lipolysis in skeletal muscle

To determine the relationship between circulating metabolic fuels and their local concentrations in peripheral tissues we measured glycerol, glucose, and amino acids by microdialysis in muscle and adipose interstitium of 10 fasted, nonobese human subjects during (a) baseline, (b) euglycemic hyperinsulinemia (3 mU/kg per min for 3 h) and, (c) local norepinephrine reuptake blockade (NOR). At baseline, interstitial glycerol was strikingly higher (P < 0.0001) in muscle (3710 microM) and adipose tissue (2760 microM) compared with plasma (87 microM), whereas interstitial glucose (muscle 3.3, fat 3.6 mM) was lower (P < 0.01) than plasma levels (4.8 mM). Taurine, glutamine, and alanine levels were higher in muscle than in adipose or plasma (P < 0.05). Euglycemic hyperinsulinemia did not affect interstitial glucose, but induced a fall in plasma glycerol and amino acids paralleled by similar changes in the interstitium of both tissues. Local NOR provoked a fivefold increase in glycerol (P < 0.001) and twofold increase in norepinephrine (P < 0.01) in both muscle and adipose tissues. To conclude, interstitial substrate levels in human skeletal muscle and adipose tissue differ substantially from those in the circulation and this disparity is most pronounced for glycerol which is raised in muscle as well as adipose tissue. In muscle, insulin suppressed and NOR increased interstitial glycerol concentrations. Our data suggest unexpectedly high rates of intramuscular lipolysis in humans that may play an important role in fuel metabolism.     

Application of muscle microdialysis to evaluate the concentrations of the fluoroquinolones pazufloxacin and ofloxacin in the tissue interstitial fluids of rats

Muscle microdialysis has been used to determine the unbound concentrations of the fluoroquinolones, pazufloxacin and ofloxacin, in tissue interstitial fluids (Cisf,u) of rats under steady state conditions. Cisf,u was estimated from the concentration in dialysate and the in-vitro permeability rate constant by the extrapolation method based on the clearance concept. Paper-disks were inserted under the abdominal skin of rats, and the drug concentrations in the fluids penetrating into the disks (Cdisk) were measured and compared with Cisf,u. The Cisf,u of pazufloxacin and ofloxacin in muscle were close to their unbound concentrations in the venous plasma; these were 75.3% and 77.1%, respectively, of the total concentrations in plasma at the steady state. The Cdisk of pazufloxacin and ofloxacin were also close to their Cisf,u. These results indicate that the unbound concentrations of the fluoroquinolones in the tissue interstitial fluids were the same as those in the venous plasma. The disk insertion technique seems to be useful for evaluating drug concentrations in tissue interstitial fluid.     

Brain-plasma distribution of free and total benzodiazepines in dogs physically dependent on different doses of diazepam

Steady-state levels of oxazepam (OX), nordiazepam (ND), and diazepam (DZ) in plasma, brain tissue, cerebrospinal fluid (CSF), and intracranial microdialysis perfusate were determined in dogs dependent on 0.56, 4.5, 9, and 36 mg/kg per day of DZ. There was a linear relationship between the total plasma and brain levels of DZ, ND, and OX and the chronic dose of DZ. Levels of free benzodiazepines in plasma and CSF and levels in microdialysis perfusates from plasma and brain were significantly correlated. With increasing dependence on DZ there was progressively more free ND and OX and less free DZ in plasma, CSF, and brain. There was a correlation between several signs of precipitated abstinence and free ND in the brain interstitial fluid, whereas convulsions emerged only when free metabolites exceeded free DZ. The changes in contribution of free DZ, ND, and OX to the overall levels of benzodiazepines present in the CNS may explain differences in signs of abstinence for different levels of dependence on DZ.     

Effects of oestradiol-18beta, hypophysectomy and age on cytoplasmic oestradiol-17beta receptor sites in rat testis interstitial tissue

After administration of oestradiol-17beta to intact mature and immature rats, a decrease in the testicular concentration of specific oestradiol-binding sites was observed within 1 h. The binding capacity was replenished starting about 3 h after oestradiol administration and after 5 h the oestrogen receptor level had returned to control values. Exposure of intact animals to oestradiol-17beta for longer periods (up to 24 h) did not result in an increase of receptor levels in testicular cytosol. Mature animals which were hypophysectomized for periods of up to 10 days did not show a significant change in the number of specific oestradiol-binding sites in either total testicular tissue or dissected interstitial tissue. At 15 days or longer periods after hypophysectomy, an apparent increase in receptor concentrations in total testicular cytosol was observed due to a relative increase in the amount of interstitial tissue. A specific oestradiol-binding protein is present in plasma of immature male rats aged less than 30 days. This plasma protein could also be demonstrated in the cytosol of testes of immature rats. In contrast to the cytosol receptor, which shows a moderate affinity for diethylstilbestrol (DES), the plasma protein did not bind DES. The sedimentation values of the plasma protein and the oestradiol receptor were 4 S and 8 S respectively. These differences in characteristics made it possible to demonstrate the presence of the oestradiol receptor in addition to the binding protein in testicular cytosol of rats from 14 days of age onwards. The nuclear receptor for oestradiol-17beta could be demonstrated after incubation of testicular tissue of rats from 4 days of age onwards.     

The binding of cyclosporin A to human plasma: an in vitro microdialysis study

PURPOSE: The human plasma binding of cyclosporin A was studied in vitro using the technique of microdialysis. The effect of temperature on the overall binding interaction between cyclosporin A and human plasma was also investigated. METHODS: Flow-through loop-type microdialysis probes were constructed from fused silica tubing and regenerated cellulose tubing with a MWCO of 13000 daltons. Probes were perfused with phosphate buffer (0.5 microliters/min) and the concentration of 3H-cyclosporin A in the well-mixed medium (plasma or buffer) was 1200 ng/ml. Relative recoveries of cyclosporin A from plasma or buffer were determined for each probe by separate experiments to measure the solute gain or loss with reference to the perfusate. RESULTS: Recoveries determined by loss were significantly greater than those determined by gain and in each case temperature dependent, with higher recoveries at higher temperatures. The plasma free fraction of cyclosporin A calculated from the recovery data and the perfusate to plasma concentration ratios was dependent on temperature in a log-linear fashion. Mean +/- s.d. plasma free fractions expressed in percent were 33.5 +/- 4.6, 17.9 +/- 3.6, 6.2 +/- 0.8, 3.0 +/- 0.6, and 1.5 +/- 0.2 at temperatures of 4, 10, 20, 30, and 37 degrees C, respectively. Assuming that the enthalpy of binding is constant over the temperature range studied and pseudo-first order conditions exist, the binding reaction at these temperatures was spontaneous, endothermic (delta H = 74.0 kJ/mole), and entropically driven (delta S = 0.274 kJ/mole/deg). CONCLUSIONS: These results show that the free fraction of cyclosporin A in human plasma is dependent on temperature with the fraction unbound decreasing with temperature in the range of 4 to 37 degrees C. The thermodynamic parameters for the binding of cyclosporin A to plasma components indicate that the reaction is a spontaneous endothermic reaction that is mainly entropy driven, similar to the partitioning of lipophilic molecules from an aqueous to a hydrophobic phase. Moreover, these results show that microdialysis is a feasible method to determine the binding interactions between plasma and cyclosporin A, which indicates the method may be suitable for other difficult binding studies where the solutes have nonspecific binding to separation devices.     

Analysis of ceftriaxone and ceftazidime distribution in cerebrospinal fluid of and cerebral extracellular space in awake rats by in vivo microdialysis

In vivo microdialysis was used to estimate the extracellular concentrations of ceftazidime and ceftriaxone, two expanded-spectrum cephalosporins commonly used in the treatment of bacterial meningitis, in two brain regions (the right corpus striatum and the left lateral ventricle_ of awake, freely moving rats. Antibiotics were administered by constant intravenous infusion at 18 mg/h until steady-state levels were reached. Ceftriaxone levels measured at the steady state in the extracellular space of the corpus striatum (0.80 +/- 0.17 micrograms/ml) were statistically equivalent to those obtained in the cerebrospinal fluid of the lateral ventricle (0.71 +/- 0.15 micrograms/ml). The ratios of these levels in the brain to the steady-state levels in plasma were 0.5 +/- 0.1% for both regions. The postinfusion concentrations of ceftriaxone in the brain declined monoexponentially, with an elimination half-life similar to that obtained in plasma. However, the mean antibiotic concentration of ceftazidime in the striatum (2.2 +/- 0.4 micrograms/ml) was lower (P < 0.001) than that in the lateral ventricle (3.8 +/- 0.5% and 4.0 +/- 1.8%, respectively) were higher than those obtained with ceftriaxone. Moreover, the half-life of ceftazidime elimination from plasma was lower than that obtained in the two brain regions. It was concluded that the in vivo microdialysis technique yields useful data on antibiotic distribution in the extracellular space of the brain, that the distribution may not be homogeneous, and that the decay of postinfusion concentrations in the brain may be different from the decay of postinfusion concentrations in plasma.     

Analysis of microdialysates from cancer patients by capillary electrophoresis

Microdialysis (MD) is an innovative clinical technique for measuring interstitial tissue pharmacokinetics and plasma-to-tissue transfer rates of drugs in humans. However, microdialysis requires the availability of specialized analytical techniques. Capillary electrophoresis (CE), which enables concentration measurements of small volume samples, theoretically constitutes an ideal analytical technique for measuring drug concentrations in microdialysates. In the present experiments, we aimed at assessing the potential utility and limitations of CE for analysis of microdialysates in a clinical situation. Microdialysates were obtained from primary breast cancer patients who received chemotherapy including 5-fluorouracil (5-FU; 600 mg/m2). Subsequently, 5-FU concentrations were measured in tumor - and subcutaneous adipose tissue - microdialysates by CE. By combining MD and CE, complete time versus concentrations profiles could be obtained for 5-FU in the interstitial tumor space and important clinical questions could be addressed. We conclude that the combination of MD and CE leads to important and previously inaccessible information about the drug distribution process in a clinical setting.     

Circumcision ceremonies at the Ottoman palace

Clinical anti-tumour efficacy of anti-cancer drugs is a function of dose intensity, i.e. the concentration--time profile in tumour tissue. Hence, information on drug concentration profiles in tumours is of critical importance but appropriate methods for measurement are lacking. The aim of the present study was to obtain, by microdialysis sampling, concentration--time profiles in a solid tumour (melanoma) of a model anti-cancer drug, carboplatin, and thereby to assess the scope of microdialysis for tumour pharmacokinetic studies in man. Six patients with cutaneous melanoma metastases at the extremities or body trunk, scheduled to receive carboplatin (400 mg m-2 i.v.) were studied. Carboplatin concentrations were monitored in serum, intratumoral and subcutaneous tissue. Calibration of the microdialysis probes was carried out in vitro and in vivo with use of the retrodialysis method. Complete carboplatin concentration vs time profiles in tumour and subcutaneous tissue were obtained. Major pharmacokinetic parameters (maximum concentration, time to maximum concentration, area under the curve, elimination half-life) were calculated for tissues and tumour/serum concentration ratios for carboplatin were derived. Mean free concentrations of carboplatin in cutaneous melanoma metastases reached only about 50-60% of total serum levels; maximal intratumoral concentrations were 7.6 (+/-2.0; s.e.m.) microgram/ml, mean concentrations in subcutaneous tissue were similar to those in tumour. The present study demonstrates that microdialysis is a novel tool for measuring drug concentrations in solid tumours in humans in vivo and appears to be a valuable addition for pharmacokinetic/pharmacodynamic studies in oncology.     

Intracranial and spinal dissemination of an ACTH secreting pituitary neoplasia. Case report and review of the literature

Smoking is associated with insulin resistance, dyslipidaemia and markers of the insulin resistance syndrome. This study investigated adipose tissue lipolysis in situ by subcutaneous microdialysis twice in 10 healthy, male smokers after smoking four cigarettes over 2 h and after the administration of an equal amount of nicotine given as nasal spray (NNS). Glucose and insulin levels, in situ lipolysis and adipose tissue blood flow were studied in the post-absorptive state and after a 75-g oral glucose tolerance test (OGTT). Post-absorptively, acute smoking and NNS increased neither subcutaneous adipose tissue glycerol production nor plasma free fatty acid (FFA) or glycerol levels. After the OGTT, plasma insulin and lactate levels were significantly higher after smoking, whereas FFA levels were higher after NNS. Normal smoking or the administration of a normal dose of NNS caused only minor metabolic changes. Thus, it does not seem likely that increased lipolysis is an important contributor to the dyslipidaemia seen in smokers.     

Theoretical analysis of the binding of salicylate by human serum albumin: the relationship between free and bound drug and therapeutic levels

The binding of salicylates by human serum albumin was analyzed by use of a computer program using previously published association constants and binding capacities for the two sets of binding sites on the protein. The analysis consisted of computing free and bound salicylates for a range of therapeutic and toxic concentration from 181 to 7246 mumole/L (25 to 1000 mg/L). At low and therapeutic levels the total amount of bound drug would exceed the amount of free drug. At higher levels, which included therapeutic and toxic ranges, the amount of free drug plasma, up to 2000 mumole/L the high affinity sites (Site 1), would bind most of the drug, but as the concentration of drug increased this site would approach saturation and the low affinity Site 2 would bind increasing amounts of salicylate. At high salicylate levels the amount of drug bound by the low affinity sites. Computation also showed that when the total amount of protein in the analysis was reduced, from 5, 4, 3, to 2 gm%, as in hypoalbuminemia, the total amount of drug bound by the protein would decrease and the quantity of free drug would increase. The amount of drug bound by each of the two sets of sites also fell as the concentration of protein decreased. Some of the possible clinical implications of these findings are discussed.     

Pharmacokinetics and extracellular distribution to blood, brain, and muscle of alovudine (3'-fluorothymidine) and zidovudine in the rat studied by microdialysis

Microdialysis was applied to sample the free drug concentration in the extracellular fluid in brain, muscle, and blood of rats given alovudine (n = 6) (3'-fluorothymidine) or zidovudine (n = 5) (25 mg/kg s.c.). Alovudine and zidovudine were analyzed by means of high performance liquid chromatography (HPLC) with UV detection. The assay for zidovudine was validated by a radioimmunoassay. In addition, the plasma protein binding of the drugs was measured by microdialysis in vitro. The concentrations attained in blood and muscle were similar for each drug, with a Cmax of 57 microM (blood) and 54 microM (muscle) for alovudine and 38 and 46 microM, respectively, for zidovudine. In contrast the Cmax in brain was 8 microM for alovudine and 4 microM for zidovudine. The peak concentration was attained 20-40 min after injection in blood and muscle and 40-60 min after injection in the brain. The half-lives of zidovudine in both blood and muscle were 37 min and in brain 69 min. For alovudine the corresponding half-lives were significantly longer: 61, 58, and 105 min, respectively. The ratio of the AUC0-180 brain/blood was 0.257 for alovudine and 0.186 for zidovudine. The plasma protein binding of zidovudine was 10%, while alovudine was virtually unbound.     

Vascular anatomy at the ureteropelvic junction

In roosters, fertility peaks to 96% at 32 weeks, shortly after sexual maturation, and then declines rapidly to 68% at 70 weeks and to less than 10% at 110 weeks, as a result of intratesticular retention of spermatozoa. The reduction in fertility is associated with functional structural changes of the interstitial tissue, reflected in decreased plasma androgen levels from 2.7 ng/ml at 32 weeks to less than 0.5 ng/ml at 110 weeks. In high fertility roosters, the interstitial tissue is tightly packed with Leydig cells, which contain relatively large amounts of rough endoplasmic reticulum and lipid droplets, both related to androgen synthesis. In the old rooster, which has a low fertility, the interstitial tissue contains only occasional Leydig cells within an enlarged intercellular space. These Leydig cells contain small amounts of endoplasmic reticulum, mainly rough, and there are low plasma androgen levels. It is concluded that differentiation of roosters' interstitial tissue is reflected by plasma levels of androgen. This, in turn, is related to the mechanism of spermatozoa release from Sertoli cells and, consequently, with the level of fertility.     

A novel dialysis procedure measuring free Zn2+ in bovine milk and plasma

A dialysis method was developed for measuring free Zn2+ concentration in plasma and milk for determination of the electrochemical gradient for Zn2+ across the mammary gland. This method used the zinc content of casein after dialysis as the metal ion indicator because zinc in the calcium phosphate-citrate complex inside casein micelles is dependent on the free Zn2+ concentration of an associated aqueous phase. Zinc, calcium and magnesium distribution in milk confirmed the high zinc binding by bovine casein. Zinc in the casein, dialyzed against standards or unknowns, was measured by atomic absorption spectrophotometry. Average free Zn2+ concentrations measured by the dialysis method in plasma and milk of five cows were 0.141 and 0.331 nmol/L, respectively. The equilibrium potential of Zn2+ ions across the mammary epithelium, calculated from free Zn2+ concentrations in blood and milk measured by the dialysis method, was -11.4 mV, consistent with the mammary electrical potentials noted in previous studies. Therefore, no electrochemical gradient for zinc between the two compartments was apparent, and it is not necessary to invoke an active transport mechanism in the mammary gland to explain the higher zinc concentration in milk than in plasma of most species.     

Ultrasonic liposculpturing: extrapolations from the analysis of in vivo sonicated adipose tissue

BACKGROUND: A local renin-angiotensin system in the heart is often invoked to explain the beneficial effects of ACE inhibitors in heart failure. The heart, however, produces little or no renin under normal conditions. METHODS AND RESULTS: We compared the cardiac tissue levels of renin-angiotensin system components in 10 potential heart donors who died of noncardiac disorders and 10 subjects with dilated cardiomyopathy (DCM) who underwent cardiac transplantation. Cardiac levels of renin and prorenin in DCM patients were higher than in the donors. The cardiac and plasma levels of renin in DCM were positively correlated, and extrapolation of the regression line to normal plasma levels yielded a tissue level close to that measured in the donor hearts. The cardiac tissue-to-plasma concentration (T/P) ratios for renin and prorenin were threefold the ratio for albumin, which indicates that the tissue levels were too high to be accounted for by admixture with blood and diffusion into the interstitial fluid. Cell membranes from porcine cardiac tissue bound porcine renin with high affinity. The T/P ratio for ACE, which is membrane bound, was fivefold the ratio for albumin. Cardiac angiotensinogen was lower in DCM patients than in the donors, and its T/P ratio was half that for albumin, which is compatible with substrate consumption by cardiac renin. CONCLUSIONS: These data in patients with heart failure support the concept of local angiotensin production in the heart by renin that is taken up from the circulation. Membrane binding may be part of the uptake process.     

Why are circulating concentrations of endothelin-1 so low?

Physiological and pathophysiological roles of endothelins are still unclear. One reason is that circulating endothelin levels in normal and pathological states are much lower than the concentrations necessary to elicit contractions in vitro. It is usually assumed that endothelin accumulates in diseased tissues and that, because of its degradation, only a small fraction of it reaches the systemic circulation. Such a hypothesis does not fit with recent observations showing that low circulating endothelin levels may be active. We show here that most of the current inferences about the actions of endothelin assume that the peptide acts in the vessel wall under conditions known as non-stoichiometric binding conditions, that is, under conditions in which the receptor concentration in tissues ([Ro]) is smaller than the equilibrium dissociation constant of endothelin receptor complexes (Kd). Under stoichiometric binding conditions (defined by the condition [Ro] > Kd), most ligand molecules are bound to receptors and cannot be present in a free form. Estimates of [Ro] and Kd from the literature suggests that in vivo endothelin probably binds stoichiometrically to its receptors. Under this condition, most of tissue endothelin is probably bound to receptors. It is therefore suggested that plasma endothelin levels are low probably because tissue free endothelin levels are low, and this is not inconsistent with the presence of high tissue levels of active (that is, bound) endothelin. When the topology of the vessels with respect to the site of production (or of delivery) of endothelin is considered, stoichiometric binding may also account for the higher sensitivity to Et-1 of in vivo preparations. It also suggests that autocrine and paracrine actions of Et-1 are favoured at low and high secretory rates respectively, thus providing an explanation for the dual (vasodilator and vasoconstricting) actions of endothelin. Finally, the stoichiometric binding model predicts that functional receptors also act as clearance receptors and provides an explanation for the observation that antagonists of endothelin receptors are also clearance antagonists.     

Quantitative microdialysis of ethanol in rat striatum

We have applied a steady-state theory of microdialysis to characterize the diffusion of ethanol through a microdialysis membrane and through rat striatum. Quantitative characterization required measurement of in vitro and in vivo extraction fractions for ethanol and determination of the clearance of ethanol from brain tissue during steady-state perfusion through a microdialysis probe. Extraction fraction of ethanol was determined in vitro by perfusing a known concentration of ethanol through probes immersed in water at 37 degrees C with stirring. The in vitro extraction fraction yielded a probe permeability value of 0.046 +/- 0.004 cm/min that is comparable with an estimate from published measurements for similar dialysis membranes. The in vivo extraction fraction was determined for probes placed in the striatum. Clearance of ethanol and a brain slice concentration profile of ethanol were determined by measurement of the amount of ethanol remaining in the brain tissue during steady-state perfusion of the probe. Steady state was achieved within 10 min after beginning the ethanol perfusion in vivo, and the extraction fraction was not altered by sedation of the rat with pentobarbital. The tissue concentration profile was symmetrical around the probe track, and ethanol was detected 1 mm from the probe. The experimental clearance rate constant value obtained for ethanol (2.0 +/- 0.3 min(-1)) was higher than that expected for removal solely by loss to the blood. The tissue diffusivity for ethanol, Dt, derived from the experimental measurements was 1.2 +/- 0.2 x 10(-5) cm2/sec. This value is greater than expected for interstitial diffusion, suggesting a substantial contribution by transcellular diffusion of ethanol as well. The predicted tissue concentration profile had a higher peak value and did not extend into the tissue (0.5 mm) as much as the experimental profile (1 mm), although there was reasonable agreement between experiment and theory. Our quantitative characterization of the microdialysis behavior of ethanol in brain provides a framework for interpretation of brain microdialysis experiments using ethanol by supplying, inter alia, a means for estimating the ethanol concentration achieved in the tissue volume being sampled by the probe.