Transmembrane fluxes of potassium in dog kidney slices. A quantitation of the effect of warm ischemia

The effect of warm ischemia on the transmembrane transport of potassium in dog kidney slices was studied by measurement of the uptake of 42K. The requirement for steady-state conditions concerning the intracellular potassium concentration was thereby studied. The total potassium content in the slices was found to be constant between 120 and 180 min incubation at both 25 and 37 degrees C. The cell water calculated from the total tissue water and 14C-inulin space in the dog kidney slices amounted to 38 ml-100 g wet weight-1 at 37 degrees C and 45 ml-100 g wet weight-1 at 25 degrees C and was found to remain constant for the incubation interval 120--180 min. The major part of the tissue uptake of 42K could be described by one single mono-exponential function under these conditions. The transmembrane influx at 37 degrees C calculated by using a modified Keynes formula amounted to 1.70 mmol K+-kg wet weight-1-min-1 after no warm ischemia and to 0.89 mmol K+-kg wet weight-1-min-1 after 2 h warm ischemia. The corresponding values for incubation at 25 degrees C were 1.26 and 0.77 mmol K+-kg wet weight-1-min-1, respectively. In the slices incubated at 25 degrees C, the potassium content was higher and the sodium content lower than in slices incubated at 37 degrees C.     

Urinary acidification: CO2 transport by the rabbit proximal straight tubule

Proximal straight tubules from rabbit kidneys were perfused in vitro in order to study transport of bicarbonate. Total CO2 content was measured in perfused and collected tubule fluid, using microcalorimetry. When the initial perfusate and bath contained 25 mM bicarbonate, the concentration of total CO2 decreased in the collected tubule fluid, indicating net reabsorption of bicarbonate. When the initial perfusate contained no bicarbonate and the bath contained 25 mM bicarbonate, total CO2 appeared in the collected tubule fluid. The rate at which total CO2 appeared in the tubule fluid was rapid, indicating a high permeability. Proximal straight tubules from superficial and juxtamedullary nephrons were compared and found to differ in permeability to CO2 and in transport rate. This functional heterogeneity may affect urinary acidification when there is redistribution of renal blood flow.     

Hereditary variation in uric acid transport by avian kidney slices

Uric acid transport in renal cortical slices from a selected line of hyperuricemic chickens was investigated. Slices from the hyperuricemic (HUA) line accumulated less than half as much uric acid as slices from a control (LUA) line when uric acid in the medium varied from 0.01 to 5 mM. Uric acid uptake by both lines increased as the uric acid concentration in the medium was raised from 0.1 to 0.5 mM, but was markedly inhibited in the HUA line at 3-5 mM. Omission of sodium or potassium from the incubation medium inhibited uric acid uptake by slices from both lines. Ouabain inhibited uric acid uptake in the LUA line. The sodium and potassium requirements for initiation of uric acid uptake were higher, and the potassium requirement for maximal uptake was lower, for slices of the HUA line. No genetic differences in potassium or sodium contents of slices were observed when the potassium content of the incubation medium was altered or when the medium contained ouabain. These studies indicate that hereditary hyperuricemia in chickens may be due to a qualitative change in renal uric acid transport which involves the interaction of cations in the transport process.     

Culture of dialysis fluids on nutrient-rich media for short periods at elevated temperatures underestimate microbial contamination

Recommended culture methods for monitoring bacterial contamination of H2O, dialysate and bicarbonate concentrate in dialysis centers in the USA involves culturing these fluids for 48 h at 37 degrees C. A variety of media and commercial culture methods are accepted for monitoring these fluids. Over a 3-month a comparison was made between an acceptable culture method, tryptic soy agar (TSA) employing the pour plate (PP) technique at 37 degrees C for 48 h, and PP cultures on standard methods agar (SMA) and R2A agar, incubated at ambient temperature (23 degrees C) for 48, 72, 168 h. Increases in the colony counts over time occurred for all three fluids. However, counts wee greater on SMA and R2A than on TSA. The increases over the standard 48-hour TSA cultures ranged as high as 10(4) times for 23 degrees C cultures at 7 days of incubation. Endotoxin levels even in the most contaminated samples were found to be below the acceptable 5 EU/ml recommended for reprocessor water. Bacterial colonies that appeared at 48, 72 and 168 h were isolated and identified. Pseudomonas, Moraxella, Acinetobacter and CDC group VI C-2 were among some of the common bacteria isolated. This study indicates that the media utilized, the time and temperature of incubation may result in a significant underestimation of the bacterial population of water and dialysis fluids, thus potentially placing the patient at a higher risk.     

Increased resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol

L-cell fibroblast cultures were treated with certain oxygenated derivatives of cholesterol which are known to inhibit cholesterol biosynthesis in mammalian cells. After incubation in the presence of 20-alpha-hydroxycholesterol or 25-hydroxycholesterol for 18 h, the cells became increasingly resistant to streptolysin O. Maximum resistance to toxin was obtained by incubation for 48 h in 0.5 microgram of 20-alpha-hydroxycholesterol or 0.25 microgram of 25-hydroxycholesterol per ml; under these conditions, the cells were 10 to 50 times more resistant than were untreated controls. The ability of the hydroxycholesterol compounds to render the cells resistant was related to the age of the cultures. Maximum protection was found when more sparsely populated cultures were treated with 25-hydroxycholesterol. Older, heavily populated cultures could not be protected even with the high concentrations of 25-hydroxycholesterol. In contrast to control cultures, most of the toxin activity remained in the medium after being incubated with hydroxycholesterol-treated cultures. The results indicate that less toxin binds to the resistant cells and suggest that a reduction in membrane cholesterol content may account for resistance to streptolysin O.     

Triazolam-induced modulation of muscarinic acetylcholine receptor in living brain slices as revealed by a new positron-based imaging technique

The effect of triazolam, a potent benzodiazepine (BZ) agonist, on muscarinic acetylcholinergic receptor (mAChR) binding was investigated in living brain slices by use of a novel positron-based imaging technique. Fresh rat brain slices were incubated with [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a mAChR antagonist, in oxygenated Krebs-Ringer solution at 37 degrees C. During incubation, time-resolved imaging of [11C]NMPB binding in the slices was constructed on the storage phosphor screens. Addition of triazolam (1 microM) plus muscimol (30 microM), a GABA(A) receptor agonist, to the incubation mixture decreased the specific binding of [11C]NMPB. Ro15-1788, a BZ receptor antagonist, prevented this effect, indicating that the effect was exerted through the GABA(A)/BZ receptor complex. These results demonstrated that stimulation of the GABA(A)/BZ receptor lowers the affinity of the mAChR for its ligand, which may underlie the BZ-induced amnesia, a serious clinical side effect of BZ. No such effect in the P2-fraction instead implies that the integrity of the neuronal cells and/or their environment is prerequisite for the modulation of mAChR by GABA(A)/BZ stimulation.     

Synthesis of 17beta-estradiol by isolated ovarian tissues of the pregnant rat: aromatization in the corpus luteum

Corpora lutea from pregnant rats were incubated to determine their ability to produce 17beta-estradiol and to aromatize testosterone in vitro. Corpora lutea and non-luteal ovarian tissues were removed from rats on days 7, 15, and 22 of pregnancy, and these tissues were immediately frozen or incubated separately in medium 199 at 37 C in an atmosphere of 95% O2-5% CO2 for 4 h. 17Beta-Estradiol in tissue and medium were quantified by a highly specific radioimmunoassay. The estradiol content ivnariably increased in non-luteal tissues during incubation, while it decreased or remained the same in incubated corpora lutea. The synthesis in non-luteal tissues, which was 18 to 400-fold greter. The incubation of corpora lutea (5 to 25 mg of tissue) with testosterone (200 ng) on days 7, 15, and 22 of pregnancy resulted in a mean accumulation of 17beta-estradiol in medium of 2.5 x 103 pg/mg tissue, compared with a mean value of 6 pg/mg for luteal tissue removed from the same ovaries and incubated without testosterone. The incubation of corpora lutea from 15-day pregnant rats with (7alpha-3H)-testosterone resulted in 15% conversion to presumptive (7alpha-3H)17beta-estradiol, which was isolated identically to estradiol isolated for radioimmunoassay. Recrystallization to constant specific activity revealed a high degree of radiochemical purity (75%) of the isolated (3H)estradiol. Rat diaphragm muscle and rabbit corpora lutea did not aromatize testosterone to 17beta-estradiol in amounts detectable by radioirpora lutea in vitro is virtually diol by non-luteal ovarian tissues. However,the corpora lutea show a striking capacity to aromatize testosterone, which might explain the high estradiol content of the rat corpora lutea during pregnancy. The physiological significance of this aromatizing system and of 17beta-estradiol in the corpus luteum is unknown but may be related to the luteotropic action of estradiol in the pregnant rat.     

Disposition of 5-hydroxytryptamine in lungs of developing rats

The pulmonary vasculature has been implicated in the clearance of several vasoactive peptides, prostaglandins, and biogenic amines from the circulation. In view of the age-related differences in the metabolism of angiotensin by intact lungs, it was of interest to examine the maturation of 5-hydroxytryptamine (5-HT) disposition by isolated perfused lungs. Lungs from newborn and adult rats were perfused with Krebs bicarbonate buffer containing 0.1 microM 5-[14C]HT and samples of the effluent medium collected and analyzed for 5-HT and metabolite. Adult lungs removed and a greater fraction of perfused 5-HT than did lungs from 7-day-old rats. No age-related difference in monoamine oxidase (MAO) activity was observed; however, lung slices from adult rats incubated with 5-[14C]HT accumulated radiolabel at a greater rate than did slices from lungs of 7-day-old rats. The age-related difference in 5-HT clearance by intact lungs may be attributable to a relative deficiency in the facilitated transport process for 5-HT in newborns and may reflect a general functional maturation of processes associated with pulmonary endothelial cell membranes.     

Cold stable microtubules in brain studied in fractions and slices

Microtubules were shown to remain intact in brain slices and subfractions maintained at 0 degrees C for 1 h. Under the same conditions, microtubules isolated from brain by warm assembly-cold disassembly methods, disassemble into their constituent subunit proteins. No selective depletions of microtubules were seen when brain slices were incubated in homogenizing buffer at either 0 degrees C or 37 degrees C. The response of native microtubules in brain slices in incubation in other solutions showed that their properties were otherwise the same as those of assembled microtubules. The separated alpha and beta subunits of isolated cold labile and cold stable microtubules were compared by electrophoresis and isoelectric focusing and were shown to possess the same mobilities. The results suggest that native microtubules are temperature insensitive and that isolated microtubules are assembled from pre-existing pools of subunit proteins. The results further suggest that native microtubules possess a factor, lacking in isolated assembled microtubules, which confers temperature stability on the former.     

The proliferation and differentiation of neonatal epidermal melanocytes in F1 hairless mice of HR-1 x HR/De in serum-free culture

Cellular uptake of neutral red dye (NR) is currently used as an indirect measure of viable cells in cultures. We used E-63 rat skeletal muscle cells to identify causes of NR assay variability and to develop modifications that substantially reduce it. Three methods of NR preparation and/or addition to cells were used. When NR medium was prepared, incubated overnight, and filtered to remove precipitates, the amount of dye precipitated varied greatly. Coefficients of variation (CVs) in NR uptake were greater than 25% between assays. Higher NR concentrations, longer incubation times, increased pH, and decreased temperature promoted NR precipitation in media. NR media prepared and filtered just prior to use or direct addition of prefiltered NR stock solution to cell cultures resulted in much smaller CVs between assays. NR was cytotoxic to E-63 rat muscle and primary quail myoblasts in a time- and concentration-dependent manner. NR exposure to E-63 cells for greater than 1.25 and 2 hr at 157 or 127 microg/ml, respectively, was associated with swelling and rupture of lysosomes. By contrast, there was no evidence of cytotoxicity when E-63 cells were exposed to NR for 1 hr at either 127 or 157 microg/ml. Primary quail myoblasts developed lysosomal swelling and ruptured more rapidly than E-63 cells when exposed to NR at either 127 or 157 microg/ml. For confluent 10-day cultures of E-63 cells exposed to NR at 127 microg/ml for 1 hr, the CVs within assay and between assays were 3.3-3.9% and 5.1%, respectively. For similarly exposed, actively replicating 3-day cultures of E-63 cells, the CVs within and between assays were 6.2-9.6% and 2.4%, respectively. NR uptake by the E-63 cells was linear with respect to viable cell number.     

Effect of metabolic alterations on the accumulation of technetium-99m-labeled d,l-HMPAO in slices of rat cerebral cortex

It is widely recognized that the distribution of technetium-99m-labeled d,l-hexamethylpropylene amine oxime (99mTc-HMPAO) in the brain is determined by the regional blood flow. However, other factors may affect this process including the metabolism of the brain tissue. To examine this possibility we studied the effects of metabolic alterations on 99mTc-HMPAO uptake in rat brain cortex slices, with concurrent measurement of oxygen consumption (QO2). 99mTc-HMPAO uptake was determined by incubating slices of rat cerebral cortex at 37 degrees C in Krebs-Ringer phosphate glucose medium containing 99mTc-HMPAO with and without test substances. Differential gradients for 99mTc activity between the tissue and the suspending medium (T/M ratio) were derived from the equation T/M[99mTc] = counts per gram of tissue/counts per milliliter of medium. The QO2 of the brain slices was measured using a biological oxygen monitor equipped with a polarographic oxygen probe. Inhibitors affecting oxidative phosphorylation caused parallel suppression of the T/M ratio and QO2. Agents that uncouple oxidation from phosphorylation increased the QO2 and decreased the T/M ratio. Incubation of slices at 22 degrees C depressed the T/M ratio and QO2. The presence of inhibitors of oxidative phosphorylation in the incubation medium increased the release of 99mTc activity from slices that had been prelabeled with 99mTc-HMPAO. These findings suggest that the altered metabolic status of the brain tissue modulates the kinetics and net accumulation of 99mTc-HMPAO at the cellular level by either depressing uptake, increasing back-diffusion, or both.     

Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.     

Measurement of the total concentration of functional Na+, K(+)-pumps in rumen epithelium

Using the technique of vanadate-facilitated [3H]ouabain binding we have developed a simple and reliable assay for measuring the concentration of [3H]ouabain binding sites in small fresh or frozen biopsies of rumen epithelium papillae. In bovine and ovine rumen epithelium obtained from the cranio-ventral rumen sac the concentration of [3H]ouabain binding sites was 1.6-4.9 nmol g dry wt-1 (n = 32) and 3.7-5.2 nmol g dry wt-1 (n = 6), respectively. When incubated in oxygenated Krebs-Ringer bicarbonate buffer fresh biopsies of rumen epithelium maintained a high K+ and low Na+ content for at least 6 h. Na+ loading of the biopsies induced about 20-fold increase of the Na+, K(+)-pump activity based on measurement of ouabain suppressible net [86Rb+] influx. The ouabain suppressible net influx of [86Rb+] measured in Na+ loaded biopsies showed a close correlation to the [3H]ouabain binding capacity (r = 0.80, P < 0.01) and corresponded to 47 +/- 2% (n = 9) of the theoretical maximum flux rate. The ouabain suppressible net influx of K+ and [86Rb+] were linearly related (r = 0.73; P < 0.001). The net Na+ efflux was 1.21 times the net K+ influx. It is concluded that rumen epithelium has a large capacity for active Na+/K+ transport and that there is agreement between the concentration of [3H]ouabain binding sites in the epithelium and the ouabain suppressible rate of net [86Rb+] influx in Na+ loaded biopsies in spite of some uncertainty about the maximum turnover number of the Na+, K(+)-pump in rumen epithelium.     

On a possible mechanism of energy conservation in sarcoplasmic reticulum membrane

The activation of ATP equilibrium Pi exchange, ITP equilibrium Pi exchange, and the degree of phosphorylation of the membrane of sarcoplasmic reticulum vesicles by ATP, ITP, and Pi were compared under different experimental conditions. In media containing 0.1 mM CaCl2, 6 mM Pi, and 4 mM ATP, during the period of Ca2+ accumulation the rate of ATP equilibrium Pi exchange was very low and the level of membrane phosphorylation by ATP was about 10-fold higher than the level of membrane phosphorylation by Pi. When net Ca2+ accumulation ceased and the Ca2+ concentration of the assay media had fallen to less than 5 muM, the degree of membrane phosphorylation by ATP decreased 4-fold and both the level of membrane phosphorylation by Pi and the rate of ATP equilibrium Pi exchange increase 4- to 6-fold. Contrasting with these data, when ATP was replaced by ITP, the rate of ITP equilibrium Pi exchange and the level of membrane phosphorylation by Pi were already high during the period of Ca2+ accumulation and varied slightly when the Ca2+ concentration of the incubation medium decreased to less than 5 muM. During the period of Ca2+ accumulation, the degree of membrane phosphorylation by Pi varied inversely with the NTP or NDP concentration of the medium, ATP and ADP being more effective than ITP and IDP in inhibiting the membrnae phosphorylation by Pi. Leaky vesicles incubated in media containing a high Ca2+ concentration were still able to catalyze both ATP equilibrium Pi and ITP equilibrium Pi exchange. Although the membrane of leaky vesicle was amply phosphorylated by Pi in media containing 0.1 nM CaCl2 and ITP, a significant rate of ITP equilibrium Pi exchange could only be measured in Ca2+ concentrations higher than 0.5 mM. The Ca2+ concentration required for half-maximal activation of the rate of either ITP equilibrium Pi or ATP equilibrium Pi exchange in leaky vesicles was found to be in the range of 1 to 2 mM. In leaky vesicles, the apparent Km of Pi for the ITP equilibrium Pi exchange was at least 1 order of magnitude lower than for the ATP equilibrium Pi exchange.     

Effects of space flight factors at the cellular level: results of the Cytos experiment

Paramecium tetraurelia was cultivated aboard the Soviet orbital station Salyut 6. Each culture included one cell, bacterized culture medium, and two small glass tubes filled with a fixative. Cultures were kept at a low temperature before Soyouz-Salyut docking. Cultures were maintained at 25 degrees +/- 0.1 degree C in orbit and were fixed every 12 h. The space flight resulted in an increase in cell growth rate and in cell volume. Measurements of cell dry weight and total protein content favour a higher cell water content. Respective roles of cosmic rays and microgravity are discussed. Cytos results are compared to those of previous space experiments.     

Oxygen/glucose deprivation in hippocampal slices: altered intraneuronal elemental composition predicts structural and functional damage

Effects of oxygen/glucose deprivation (OGD) on subcellular elemental composition and water content were determined in nerve cell bodies from CA1 areas of rat hippocampal slices. Electron probe x-ray microanalysis was used to measure percentage water and concentrations of Na, P, K, Cl, Mg, and Ca in cytoplasm, nucleus, and mitochondria of cells exposed to normal and oxygen/glucose deficient medium. As an early (2 min) consequence of OGD, evoked synaptic potentials were lost, and K, Cl, P, and Mg concentrations decreased significantly in all morphological compartments. As exposure to in vitro OGD continued, a negative DC shift in interstitial voltage occurred ( approximately 5 min), whereas general elemental disruption worsened in cytoplasm and nucleus (5-42 min). Similar elemental changes were noted in mitochondria, except that Ca levels increased during the first 5 min of OGD and then decreased over the remaining experimental period (12-42 min). Compartmental water content decreased early (2 min), returned to control after 12 min of OGD, and then exceeded control levels at 42 min. After OGD (12 min), perfusion of hippocampal slices with control oxygenated solutions (reoxygenation) for 30 min did not restore synaptic function or improve disrupted elemental composition. Notably, reoxygenated CA1 cell compartments exhibited significantly elevated Ca levels relative to those associated with 42 min of OGD. When slices were incubated at 31 degreesC (hypothermia) during OGD/reoxygenation, neuronal dysfunction and elemental deregulation were minimal. Results show that in vitro OGD causes loss of transmembrane Na, K, and Ca gradients in CA1 neurons of hippocampal slices and that hypothermia can obtund this damaging process and preserve neuronal function.     

Effects of variations in hippocampal slice preparation protocol on the electrophysiological stability, epileptogenicity and graded hypoxia responses of CA1 neurons

Evoked, extracellularly recorded field potentials and whole-cell current-clamp recordings were used to assay the effects of variations in dissection method and incubation temperature on the electrophysiology of CA1 neurons in hippocampal slices. Slices were cut with either a vibratome or a tissue chopper, and incubated at 28-30 degrees C, room temperature (19-21 degrees C), or in cool solution (13-15 degrees C) which was allowed to passively warm to room temperature while the slices were incubating ('cold-shock', CS). Although no effects of dissection method were observed, it was found that incubation temperature had profound effects on synaptically, but not non-synaptically evoked field potentials. Cold-shocked slices, cut with either a vibratome or a tissue chopper, exhibited epileptiform and spontaneously potentiating orthodromic field potentials. Slices incubated at warmer temperatures demonstrated responses that were larger in amplitude, more stable and much less epileptiform. In response to orthodromic stimulation, CS neurons fired more action potentials than did neurons in slices incubated at room temperature. Further, CS neurons generated smaller inhibitory postsynaptic potentials. Field potential changes resulting from graded hypoxia were not significantly affected by the temperature at which the slices were incubated. These data suggest that the electrophysiology of the hippocampal slice can be altered by the methods used to prepare the tissue. This finding may account for some of the discrepancies that exist between laboratories, and serves to underscore the importance of accurately reporting detailed protocols. Further, CS hippocampal tissue may represent a novel in vitro model of epileptiform activity.     

Monooxygenation, cytochrome P450-mRNA expression and other functions in precision-cut rat liver slices

Precision-cut rat liver slices (KRUMDIECK slicer, slice thickness 200-250 microm) were incubated in rollers containing modified William's medium E at 37 degrees C for 2, 24 and 48 hrs. Protein, DNA, potassium and glutathione concentrations did not decrease during 48 hrs. Lactate dehydrogenase (LDH) leakage into the medium was relatively marked during the first 2 hrs of incubation, from the 2nd to the 48th hr LDH leakage was very low. The same is true of the release of thiobarbituric acid-reactive substances. Albumin synthesis and transport into the medium decreased to about 70% after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation rate was relatively stable up to 48 hrs, whereas testosterone hydroxylation decreased significantly without alterations of the proportions of the 7 quantified hydroxylated metabolites. After exposure of the slices to beta-naphthoflavone for 6 hrs CYP1A1-mRNA expression, measured by competitive RT-PCR, was increased by a factor of at least 1000. Precision-cut liver slices are a useful tool for the study of various hepatic functions, drug metabolism and its induction in vitro.     

Influence of oxygen and glucose on the water and ion content of swine aorta

The swelling properties of isolated swine arterial tissue have been studied to determine their effect on diffusion and hydraulic permeability measurements. Tissue potassium and sodium content were also measured to obtain an index of tissue metabolic activity. When oxygen and glucose were present in the incubation medium, a 5% decrease in tissue water content was observed over an incubation period of approximately 3.5 h. Under these conditions the tissue potassium content was higher and the sodium content was lower than when oxygen and/or glucose were omitted from the medium. When both oxygen and glucose were omitted, the potassium and sodium levels were significantly altered, suggesting a disturbance in the sodium-potassium transport system due to depletion of necessary metabolities.     

Induction of cytochrome P450 1A1 in rat liver slices by 7-ethoxycoumarin and 4-methyl-7-ethoxycoumarin

7-Ethoxycoumarin (EC) is widely used as a model substrate for monooxygenase function, its O-deethylation representing cytochrome P450 (P450) activity mainly of 1A but also of 2B isoforms. Reports on investigations of its own capacity to induce or suppress P450 activities, however, have not been found in biomedical literature. To avoid the influence of in vivo pharmacokinetics, studies can well be undertaken with liver slice incubation. Therefore in the present investigation precision-cut rat liver slices from male 43-63-day-old male HAN:Wistar outbred rats were incubated at 30 degrees C in carbogen saturated William's Medium E for 24 h. EC was added previously to final concentrations of 10, 25, 50, 75 or 100 microM. After incubation, homogenate was prepared from slices and used for model reactions (7-ethoxyresorufin O-deethylation [EROD] and 7-pentoxyresorufin O-depentylation [PROD]). EROD, indicating activities of 1A isoforms, was enhanced by incubation with EC at 25 and 50 microM to about doublefold but showed control or lower values at 75 and 100 microM. Incubation with beta-naphthoflavone in comparison led to variable increases (3-5-fold of controls). For PROD as an indicator of the phenobarbital inducible P450 isoforms 2B1 and 2B2 no enhancement was found, but a decrease by incubation with 75 and 100 microM EC. To further investigate the correlation between enzyme activity and gene expression after slice incubation, P450 1A1 mRNA content was measured by RT-PCR. Induced gene expression for 1A1 was seen with different EC concentrations to a variable extent, though not as strong as with BNF. Similar incubation with 4-methyl-7-ethoxycoumarin revealed an even stronger induction of EROD activity with maxima at about 10-32 microM, reaching BNF values. In contrast incubation with 7-benzyloxycoumarin had no evident inducing or suppressing effect, neither on EROD nor on PROD activity.