Isolated Organ Preparations Research Articles

Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress.

Fetal hypoxia is a common complication of pregnancy. It has been shown to programme cardiac and endothelial dysfunction in the offspring in adult life. However, the mechanisms via which this occurs remain elusive, precluding the identification of potential therapy. Using an integrative approach at the isolated organ, cellular and molecular levels, we tested the hypothesis that oxidative stress in the fetal heart and vasculature underlies the molecular basis via which prenatal hypoxia programmes cardiovascular dysfunction in later life. In a longitudinal study, the effects of maternal treatment of hypoxic (13% O2) pregnancy with an antioxidant on the cardiovascular system of the offspring at the end of gestation and at adulthood were studied. On day 6 of pregnancy, rats (n = 20 per group) were exposed to normoxia or hypoxia ± vitamin C. At gestational day 20, tissues were collected from 1 male fetus per litter per group (n = 10). The remaining 10 litters per group were allowed to deliver. At 4 months, tissues from 1 male adult offspring per litter per group were either perfusion fixed, frozen, or dissected for isolated organ preparations. In the fetus, hypoxic pregnancy promoted aortic thickening with enhanced nitrotyrosine staining and an increase in cardiac HSP70 expression. By adulthood, offspring of hypoxic pregnancy had markedly impaired NO-dependent relaxation in femoral resistance arteries, and increased myocardial contractility with sympathetic dominance. Maternal vitamin C prevented these effects in fetal and adult offspring of hypoxic pregnancy. The data offer insight to mechanism and thereby possible targets for intervention against developmental origins of cardiac and peripheral vascular dysfunction in offspring of risky pregnancy.

Effect of Nicorandil upon Different Guinea-pig and Rat Isolated Organ Preparations in vitro

A study of the effect of nicorandil (N-2-(hydroxyethyl)nicotinamide nitrate, CAS 65141-46-0), a potassium channel and guanylatecyclase activator, upon preparations of rat was deferens and uterus, and guinea pig ileum was performed. Nicorandil does not modify rat isolated was deferens responses to noradrenaline (norepinephrine) and potassium. The drug exerts a non-competitive antagonist effect upon rat isolated uterus response to serotonin, histamine, oxytocin, and, at high concentrations, inhibits guinea-pig isolated ileum responses to acetylcholine, histamine, 4-aminopyridine and potassium.

Studies of the toxic effects of norepinephrine on isolated cardiomyocytes of guinea-pigs.

Catecholamines have been demonstrated to possess direct cardiotoxic effects mediated by oxygen free radicals in isolated organ preparations. In order to assess direct cytotoxic properties, the influence of exogenous noradrenaline (norepinephrine, CAS 51-41-2) (10(-6) mol/l) on isolated guinea-pigs cardiomyocytes was examined, in the presence of propranolol (10(-6) mol/l) and phentolamine (10(-6) mol/l) to inhibit adrenoceptor-mediated effects. Cell viability was assessed by morphologic examination (% of striated, rod-shaped cells), before and after a treatment period of 15 and 60 min by the measurement of intracellular enzyme activities in the supernatant of the suspension (lactate dehydrogenase, creatine kinase, aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase). The proportion of viable, rod-shaped cardiomyocytes (21.6% +/- 7.6% after preparation, before starting the treatments) significantly decreased over the experimental time (p < 0.05) and, concomitantly, the activity of intracellular enzymes in the supernatant increased. There was no difference between controls and treated suspensions. Thus, there is no evidence for direct toxic effects of norepinephrine in micromolar concentration on isolated cardiomyocytes of guinea-pigs. However, cytoprotective effects by propranolol and/or phentolamine cannot be excluded in this model.

Properly substituted 1,4-dioxane nucleus favours the selective M 3 muscarinic receptor activation

Novel analogues of cis- N, N, N-trimethyl-(6-methyl-1,4-dioxan-2-yl)methanaminium iodide ( 2a) were synthesized by inserting methyl groups alternatively or simultaneously in positions 5 and 6 of the 1,4-dioxane nucleus in all combinations. Their biological profile was assessed by receptor binding assays at human muscarinic M 1–M 5 receptors stably expressed in CHO cells and by functional studies performed on classical isolated organ preparations, namely, rabbit electrically stimulated vas deferens, and guinea pig electrically stimulated left atrium, ileum, and lung strips. The results showed that the simultaneous presence of one methyl group in both positions 5 and 6 with a trans stereochemical relationship with each other (diastereomers 4 and 5) or the geminal dimethylation in position 6 (compound 8) favour the selective activation of M 3 receptors. Compounds 4, 5, and 8 might be valuable tools in the characterization of the M 3 receptor, as well as provide useful information for the design and development of novel selective M 3 antagonists.

Apelin and Its G Protein-Coupled Receptor Regulate Cardiac Development as Well as Cardiac Function

The Apelin pathway has only recently emerged as an important regulator of cardiac and vascular function, mediating adaptation to physiological stress and disease. In this issue of Developmental Cell, experiments in zebrafish convincingly show a critical role for this pathway in myocardial cell specification and heart development.

Opioid Peptides: Synthesis and Biological Activity of New Endomorphin Analogues

Syntheses are described of new endomorphin 1 and 2 peptoid–peptide hybrids in which Tyr1 and either one or both Phe3 and Phe4 have been replaced by N-substituted-glycine. The preparation is also described of two glycosylated Hyp2-endomorphin 2 analogues in which either 2,3,4,6-tetra-O-acetyl glucose or glucose are β-O-glycosidically linked to the hydroxyproline residue. The Hyp2-endomorphin sequences have also been elongate by adding a C-terminal β-alanine residue and several linear dimers have been prepared by coupling either the native peptides or the modified analogues. The cyclo endomorphin 2 has also been synthesized. Preliminary pharmacological experiments on isolated organ preparations showed that the agonist activities of both endomorphin 1 and 2 are not significantly affected by the Pro/Hyp substitution. Phe4/Nphe substitution in the endomorphin 1 reduced the potency on guinea pig ileum (GPI) by about 100 times and abolished the agonist activity on mouse vas deferens (MVD) preparation. The decrease of the agonist activity induced by modification of one phenylalanine residue only, either Phe3 or Phe4, is lower on endomorphin 2. Either modification of both Phe3 and Phe4 or glycosylation of the Hyp2-endomorphin 2 cancelled any agonist activity on both preparations. The linear peptide dimers [endomorphin 1]2, [endomorphin 2]2, [Hyp2-endomorphin 1]2, [Hyp2-endomorphin 2]2, [Hyp2-endomorphin 1-Hyp2-endomorphin 2]2 or [Hyp2-endomorphin 2-Hyp2-endomorphin 1]2, are 7–19 times less potent than endomorphin 1 on GPI and significantly less active than endomorphins 1 and 2 on MVD. The other afforded modifications significantly affected or abolished the agonist activity of the resulting endomorphin analogues on both GPI and MVD preparations.

Design, Synthesis, and Action of Oxotremorine-Related Hybrid-Type Allosteric Modulators of Muscarinic Acetylcholine Receptors

A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.

Forty years in capsaicin research for sensory pharmacology and physiology

Capsaicin, the pungent ingredient of chilli peppers has become a "hot" topic in neuroscience with yearly publications over half thousand papers. It is outlined in this survey how this exciting Hungarian research field emerged from almost complete ignorance. From the initial observation of the phenomenon of "capsaicin desensitization", a long-lasting chemoanalgesia and impairment in thermoregulation against heat, the chain of new discoveries which led to the formulation of the existence of a "capsaicin receptor" on C-polymodal nociceptors is briefly summarized. Neurogenic inflammation is mediated by these C-afferents which are supplied by the putative capsaicin receptor and were termed as "capsaicin sensitive" chemoceptive afferents. They opened new avenues in local peptidergic regulation in peripheral tissues. It has been suggested that in contrast to the classical axon reflex theory, the capsaicin-sensitive sensory system subserves a "dual sensory-efferent" function whereby initiation of afferent signals and neuropeptide release are coupled at the same nerve endings. Furthermore, in the skin at threshold stimuli which do not evoke sensation elicit already maximum efferent response as enhanced microcirculation. In isolated organ preparations large scale of new type of peptidergic capsaicin-sensitive neurogenic smooth muscle responses were revealed after the first one was described by ourselves on the guinea-pig ileum in 1978. Recently the "capsaicin receptor" has been cloned and it is now named as the "transient receptor potential vanilloid 1" (TRPV1). Hence, capsaicin research led to the discovery of the first temperature-gated ion channel gated by noxious heat, protons, vanilloids and endogenous ligands as anandamide, N-oleoyldopamine and lipoxygenase products. Another recent achievement is the discovery of a novel "unorthodox" neurohumoral regulatory mechanism mediated by somatostatin. Somatostatin released from the TRPV1-expressing nerve endings reaches the circulation and elicits systemic antiinflammatory and analgesic "sensocrine" functions with counter-regulatory influence e.g. in Freund's adjuvant-induced chronic arthritis. Nociceptors supplied by TRPV1 and sst4 somatostatin receptors has become nowadays promising targets for drug development.

D-Ala2]-Deltorphin I peptoid and retropeptoid analogues: synthesis, biological activity and conformational investigations.

The synthesis is described of a [D-Ala2]-deltorphin I peptoid analogue in which all amino acid residues have been substituted by the corresponding N-alkylglycine residues. The [D-Ala2]-deltorphin I retropeptoid was also prepared as well as [Ala1 ,D-Ala2]-deltorphin 1 and the corresponding peptoid. Structural investigations by FT-IR and fluorescence measurements were carried out on the synthetic analogues and on some [D-Ala2]-deltorphin 1 peptide-peptoid hybrids previously prepared. According to the fluorescence measurements the distance between the aromatic residues in the deltorphin I peptoid and retropeptoid is similar to that suggested for the delta- and micro-opioids, respectively. Measurements of CD in the presence of beta-cyclodextrin, and some preliminary pharmacological experiments were also performed. No dichroic bands are present in the spectrum of the [Ntyr1,D-Ala2]-deltorphin I, but an increasing dichroic effect appears in the spectra of both the deltorphin I peptoid and retropeptoid. Activity tests on isolated organ preparations showed that the modifications made produced a dramatic decrease in the agonistic activity of the synthetic derivatives.

Molecular anatomy of the cerebral microvessels in the isolated guinea-pig brain

Isolated organ preparations represent valuable models for biomedical research, provided that the functional and morphological integrity of vascular and parenchymal compartments is preserved. In this investigation, we have studied the molecular organization of the cerebral microvessels in the isolated guinea-pig brain maintained in vitro by arterial perfusion, a preparation previously proposed as a model of blood–brain barrier (BBB). Using lectin cytochemistry and immunohistochemistry, we examined the microvasculature of the cerebral cortex after 5 h in vitro to assess: (a) the structure of the endothelial glycocalyx at microscopical and ultrastructural level; (b) the distribution of the junctional molecules occludin, ZO-1, PECAM-1 and vinculin; (c) the distribution of basal lamina molecules, such as collagen type IV, laminin and heparan sulfate proteoglycan. All these components of microvessel wall have been previously shown to be vulnerable to ischemic conditions and their organization could be altered in consequence of the transient hypoxia associated with the brain isolation procedure. Our observations demonstrate that the distribution pattern of the molecules considered (i) is comparable to that shown in the cerebral microvasculature of other mammals and (ii) is similar in brains maintained in vitro and in control brains perfused in situ with fixative. The complex of our observation indicates that the molecular organization of the cerebral microvessels is preserved in isolated guinea-pig brain, thus indicating that these preparations can be used to study the cerebrovascular structure and blood–brain barrier function in a variety of experimental conditions.

Capsaicin-like effects of N-arachidonoyl-dopamine in the isolated guinea pig bronchi and urinary bladder

A capsaicin-like endogenous ligand of vanilloid (VR1) receptors, N-arachidonoyl-dopamine, was recently identified in bovine and rat nervous tissue, and found to be almost as potent as capsaicin, and 5–10-fold more potent than anandamide, on these receptors, both in isolated cells and in vivo. Here we have investigated if N-arachidonoyl-dopamine also exerts other capsaicin-like effects at VR1 receptors in some isolated organ preparations. N-arachidonoyl-dopamine exerted a potent contractile response of guinea pig isolated bronchi (EC 50=12.6±1.7 μM, E max=69.2±2.4% of carbachol E max), which was blocked by pre-treatment with capsaicin or with the VR1 antagonist capsazepine, as well as by a combination of tachykinin NK1 and NK2 receptor antagonists. In this assay, N-arachidonoyl-dopamine was less and more potent and/or efficacious than capsaicin (EC 50=40.0 nM; E max=93.5%) and anandamide (EC 50=15.2 μM, E max=38.0%), respectively. Unlike capsaicin and anandamide, forskolin or ethanol did not enhance N-arachidonoyl-dopamine effect in this preparation, whereas epithelial denudation resulted in a 2.5-fold increase in potency without affecting the efficacy. N-arachidonoyl-dopamine also contracted the isolated guinea pig urinary bladder, although in this preparation, as well as in the isolated rat urinary bladder, the potency (EC 50=3.7±0.3 and 19.9±0.1 μM) and/or efficacy ( E max=12.0±0.1% and 20.7±0.7% of carbachol E max) of the compound were significantly lower than those of both capsaicin and anandamide. These data suggest that the extent to which exogenous N-arachidonoyl-dopamine activates VR1 receptor in isolated organs is largely dependent on pharmacodynamics and bioavailability.

Novel Series of O-substituted 8-quinolines and 4-benzothiazoles as potent antagonists of the bradykinin B 2 receptors

The synthesis and the SAR study of novel O-substituted 8-quinolines and 4-benzothiazoles as highly potent non-peptide bradykinin B 2 receptor antagonists are described. Several members of this series of antagonists efficiently inhibited the BK-induced vasoconstriction on different isolated organ preparations.

Beta-blocker selectivity at cloned human beta 1- and beta 2-adrenergic receptors.

The ratio between the affinities of beta-blockers for the beta2- and beta1-receptors is often used to predict the cardioselectivity and the potential consequences of blocking beta2-receptor-mediated effects of adrenergic receptor blockers. These ratios have been traditionally determined using various in vitro models of beta2 and beta1-receptor antagonist activity, including isolated organ preparations and radioligand binding in tissues from various species. The data from these studies, while useful, are complicated by the use of different preparations, techniques, and nonhuman models. Recombinant cell lines expressing human beta2 and beta1 receptors have been developed, allowing for the direct comparison of the affinities of the beta-blockers for the beta2 and beta1 receptors under identical conditions, and allowing a precise determination of the beta1-receptor selectivity of the beta-blockers. Bisoprolol, atenolol, propranolol, betaxolol, metoprolol, carvedilol, and ICI 118, 551 were compared for their beta-receptor selectivity using membranes prepared from recombinant cells selectively expressing human beta2 and beta1 receptors. Bisoprolol was found to have the highest selectivity for the beta1 receptor, displaying a beta2/beta1 ratio of 19 (a 19-fold higher affinity for the beta3 receptor than for the beta2 receptor). Atenolol, metoprolol, and betaxolol displayed lower, selectivity for the beta1 receptor, whereas propranolol and carvedilol displayed no significant beta-adrenergic selectivity. ICI 118,55 was selective for the beta2 receptor. The equilibrium dissociation constants of the beta-blockers for the beta1 and beta2 receptors were generally similar to previously reported values. The affinity ratios were also generally similar to previously reported values.

Bioassays for NPY receptors: old and new

Classical pharmacology performed on isolated organ preparations is an essential tool for receptor characterization and classification. Basic pharmacological parameters (e.g. ED 50, ID 50, p D 2, p A 2 as measures of apparent affinities) obtained by relating the agent concentration with the biological effect are the final results of the various steps required for drug action and necessarily reflect the complex mechanisms of cell fuction. Results obtained with bioassays are therefore a useful and essential part in the assessment of endogenous systems, in the present case, the NPY family of peptides and their receptors. An attempt has been made, in the present review, to present a choice of isolated organs that may provide a starting point towards the construction of a solid classical pharmacology of receptors for NPY and congeners. Some of these organs appear to be `monoreceptor systems' (e.g. the rabbit saphenous vein) whose response is contributed by a single receptor type, others (e.g. the rat colon) are `multiple receptor systems' and their pharmacology is much too complex and requires the use of a variety of compounds from the naturally occuring peptides, to some selective agonists and when available, specific and selective antagonists. Such compounds have been utilised by us and other workers to detect specific biological responses to NPY and congeners in peripheral tissues: such responses have been quantified, carefully analysed in pharmacological terms and characterized as biological effects mediated by Y 1 (the rabbit saphenous vein), Y 2 (dog saphenous vein, rat vas deferens, rat colon), Y 4 (rat colon) and Y 5 (rabbit ileum) receptors. Compared to findings obtained with binding assays and molecular biology experiments, the results of the bioassays show very interesting similarities. Much remains however to be done in view of providing the classical pharmacological bases that are needed in the field of NPY.

WAL 2014 FU (talsaclidine): A preferentially neuron activating muscarinic agonist for the treatment of Alzheimer's disease

The functional selectivity of WAL 2014 FU with regard to stimulation of the neuronal muscarinic M1 receptor subtype in vitro and in vivo is shown in different receptor preparations, isolated organ models, whole animal testing, and finally humans. From receptor binding experiments in membrane preparations from rat tissues and from Chinese hamster ovary cells expressing human muscarinic receptor subtypes, it can be delineated that the ratio between M1 and M2 (hm1 and hm2) is shifted in favour of the M1 receptor affinity, when compared to several classic muscarinic agonists such as carbachol, arecoline, and oxotremorine. The intermediate GTP-shift of 7.5 for WAL 2014 FU in a M2 muscarinic receptor preparation (rat heart) indicates only partial agonistic activity at this subtype, carbachol (e.g., shows a shift of 51). Moreover, the ratio from agonist to antagonist receptor binding comparing the affinities using [3H]cis-methyldioxolane and [3H]N-methylscopolamine as radioligands, suggests only a partial agonist behaviour at M2 receptors, too. In functional assays in vitro, such as phosphoinositide breakdown measured in cerebral cortex slices from guinea pig brain, WAL 2014 FU is as effective (27.8% compared to carbachol 100%) as arecoline (27.6%), but is more effective than RS 86 (22.1%), oxotremorine (21.6%) or McN-A-343 (14.7%). WAL 2014 FU is about as effective as RS 86 and McN-A-343 in stimulating the secretion of N-acetyl-glucosaminidase from RBL cells transfected with the human m1 receptor subtype. Investigating the proton efflux from CHO cells expressing hm1, hm2, and hm3 muscarinic receptor with cytosensor technology WAL 2014 FU was clearly more effective at human m1 receptors (66.3% compared to carbachol 100%) than at hm3 receptors (18.6%) and showed no measurable effect at hm2 receptor expressing cells. The efficacy in isolated organ preparations and in whole animals has already been reported [Ensinger et al., 1993] and shows the functional selectivity of WAL 2014 FU quite impressively. The general receptor profile of WAL 2014 FU demonstrates a nearly specific interaction with muscarinic receptors, having only weak binding affinity for a1- and nicotinic receptors. Results from pharmacokinetic studies in rats and humans indicate high oral bioavailability (>95% in rats) and very low interindividual variation in plasma levels. Furthermore, good penetration of the blood brain barrier with 2.5- to 3-fold higher concentrations of WAL 2014 FU in brain than in plasma could be measured. The advantageous and stable pharmacokinetic profile of WAL 2014 FU and its functional M1 selectivity establish WAL 2014 FU as a promising candidate for drug treatment of Alzheimer's disease. In addition to its role for symptomatic treatment, WAL 2014 FU may also have a disease-modifying potential because of its stimulatory effects on APPs secretion. Drug Dev. Res. 40:144–157, 1997. © 1997 Wiley-Liss, Inc.

Potencies of various neurotensin-(8–13) analogs for inhibition of heat-induced edema in the anesthetized rat

Peptides of the neurotensin (NT) and xenopsin (XP) families inhibit vascular leakage in various models of tissue injury. In this study, we measured the potency of NT fragments, NT analogs and NT-(8–13) analogs for inhibition of thermal edema induced by immersion of the anesthetized rat's paw in 58°C water for 1 min. The pattern of anti-edema potencies seen with sixteen NT-(8–13) analogs correlated well with the pattern of activities obtained in binding measurements to rat brain membrane preparations and to activities in isolated organ preparations. Replacement of Tyr 11 with Trp in NT-(8–13) and Arg 8 with d-Arg resulted in an analog [ d-Arg 8, Trp 11]NT-(8–13) which was 5-times more potent than NT-(8–13). Substitution of d-Arg for Arg 8 and Arg 9 in NT-(8–13) produced analogs that retained anti-edema activity but with decreased effects on gut motility and hypotension.

Towards the physiological function of uric acid

Uric acid, or more correctly (at physiological pH values), its monoanion urate, is traditionally considered to be a metabolically inert end-product of purine metabolism in man, without any physiology value. However, this ubiquitous compound has proven to be selective antioxidant, capable especially of reaction with hydroxyl radicals and hypochlorous acid, itself being converted to innocuous products (allantoin, allantoate, glyoxylate, urea, oxalate). There is now evidence for such processes not only in vitro and in isolated organs, but also in the human lung in vivo. Urate may also serve as an oxidase cosubstrate for the enzyme cyclooxygenase. As shown for the coronary system, a major site of production of urates is the microvascular endothelium, and there is generally a net release of urate from the human myocardium in vivo. In isolated organ preparations, urate protects against reperfusion damage induced by activated granulocytes, cells known to produce a variety of radicals and oxidants. Intriguingly, urate prevents inactivation of endothelial enzymes (cyclooxygenase, angiotensin converting enzyme) and preserves the ability of the endothelium to mediate vascular dilatation in the face of oxidative stress, suggesting a particular relationship between the site of urate formation and the need for a biologically potent radical scavenger and antioxidant.

Near-infrared spectrophotometric device to measure fluid exchange in blood-perfused organs.

We describe an on-line spectrophotometric device designed to estimate the volume of transvascular fluid exchange (VE) continuously in isolated organ preparations. The device is interposed in the perfusion circuit, and VE is continuously estimated from changes in light transmission proportional to changes in perfusate hematocrit. In our perfusing circuit, hemolysis was negligible. Using hourly determinations of free hemoglobin, we estimated errors in VE due to hemolysis to be on the order of 1 ml for every hour of perfusion (expt I). When logarithms of on-line spectrophotometric signals were plotted against measured hematocrits (expt II), there was always an excellent linear correlation over a wide range of hematocrits (from 0.247 to 0.583). Finally, there was also an excellent correlation between spectrophotometric [QF(Spectr)] and gravimetric [QF(wt)] estimates of constant rates of transvascular fluid exchange in five isolated excised canine left lower pulmonary lobes, where QF(Spectr) = delta VE/delta t and QF(wt) = delta wt/delta t (expt III). QF(Spectr) = 1.035QF(wt) - 0.038 (r2 = 0.9772).

Red blood cells augment transport of reactive metabolites of monocrotaline from liver to lung in isolated and tandem liver and lung preparations

Monocrotaline (MCT) is a pyrrolizidine alkaloid that causes pulmonary hypertension in rats by mechanisms which remain largely unknown. MCT is thought to be activated in the liver to a reactive intermediate that is transported to the lung where it causes endothelial injury. Our previous pharmacokinetic work demonstrated significant sequestration of radioactivity in red blood cells (RBCs) of rats treated with [14C]MCT. To determine whether this RBC sequestration might be important in the transport of reactive MCT metabolites, we compared the effect of inclusion of RBCs in the perfusion buffer on the extent of covalent binding of [14C]MCT to rat lungs in tandem liver-lung preparations. The potential effect of RBCs in stabilizing reactive intermediates was evaluated by preperfusion of isolated liver preparations with [14C]MCT with and without RBCs, separation and washing of the RBC fraction, and subsequent (90 min later) perfusion of washed RBCs or buffer alone in isolated perfused lungs. Covalent binding to lung tissues was determined by exhaustive methanol/chloroform extractions of unbound label from homogenized lung tissue followed by scintillation counting of residual 14C. Covalent binding was expressed as picomole MCT molecular weight equivalents/mg protein. Comparison of the relative capability of these isolated organ preparations for conversion of MCT to polar metabolites was done by extraction and HPLC analysis of perfusate at the end of the experiment. Isolated livers converted 65-85% of MCT to polar metabolites compared with less than 5% conversion in the isolated lungs. Inclusion of RBCs in the buffer of tandem lung liver preparations perfused with 400 microM [14C]MCT increased the covalent binding to the lung from 97 +/- 25 (buffer alone) to 182 +/- 36 (buffer + RBC) pmol/mg protein. At the end of these perfusions, RBCs contained 1552 +/- 429 pmol/mg hemoglobin of which 333 +/- 98 pmol/mg hemoglobin resisted exhaustive solvent extraction. After 90 min at room temperature, buffer with 400 microM [14C]MCT preperfused in isolated livers resulted in covalent binding to isolated perfused lung of 0.8 +/- 0.4 pmol/mg protein while washed RBCs isolated from buffer of similar liver preperfusions preparations resulted in 53 +/- 7 pmol/mg protein bound to lung. Control groups perfused with 400 microM [14C]MCT in buffer or buffer + RBCs through isolated lungs only resulted in covalent binding of 2 +/- 1 or 1 +/- 0.6 pmol/mg protein respectively. We conclude: (1) RBCs significantly augment the transport of lung reactive MCT metabolites from the liver to the lung.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of lactate production by tracer techniques

Tracer methodology is developed for use in the study of lactate metabolism. A one-compartment model introduces the inference of lactate flux from specific activity. Two compartments distinguish between blood and tissue, and give an estimate of tissue specific activity from measurements in blood. The two-compartment model is applied directly to the problem of flux between lactate and pyruvate in a single tissue. It is shown that equilibration between lactate and pyruvate pools does not invalidate the use of tracer methodology to study lactate metabolism. Application of the two-compartment model to a perfused muscle preparation shows that both lactate and pyruvate fluxes can be measured by using tracer techniques. A three-compartment formalism is presented to resolve the controversy regarding sampling and infusion sites, with special attention given to anatomical identity of the sites. Future directions include measurement of tracer transients during tracee steady state, measurement of distribution of blood flow, and further study of isolated organ preparations.