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Incubation of intact human erythrocytes with [3H]palmitate labeled a protein with electrophoretic characteristics of the glucose transporter. This labeling occurred via a thioester linkage, since it was unaffected by organic solvent extraction, but was substantially removed as the hydroxamate upon treatment with neutral hydroxylamine. Immunoprecipitation of the labeled protein with a monoclonal antibody to the glucose transporter confirmed its identity.

We describe the synthesis of the isotopically labelled nitrone spin traps 7,9, and 10, and spin labelled cytochalasin B derivatives 15 23. We describe a new general route to proxy1 nitroxide spin labelled fatty acids, for example, 28 and 29, and the 15Nnonadeutero 1 1-proxylpalmitic acid P-OH. New types of paramagnetic liposomes derived from bis nitroxide quaternary salts 31 and 32 have been prepared. Molecules designed as molecular amplifiers such as hexaamine 35a are described. Paramagnetic centers such as a nitroxide epoxide or Gd complex 37 may be attached giving unique spin relaxers such as 35b and 35c to which an isothiocyanate reactive group may be added giving 36b and 36c. INTRODUCTION Stable nitroxide free radicals continue to play a central role in a wide variety of investigations, especially involving spin labelling and spin trapping applications. More recently, novel applications have arisen in which nitroxides are being developed as contrast-enhancing agents for magnetic resonance imaging (MRI) and as paramagnetic agents for the relatively new field of imaging by electron spin resonance (ESR) spectroscopy. In this paper we summarize several recent developments in our laboratory in the nitroxide field. I. Development of *Hand '5N-isotopically substituted nitrone spin traps organic free radicals. In this method an appropriate nitrone or nitroso compound reacts with the short lived radical intermediate producing a nitroxide with a lifetime considerably greater than that of the parent radical. Recent advances in spin trapping include the preparation of a family of pyrrolidine-I-oxide spin traps with enhanced lipophilicity and a decreased tendency toward oxidation3 and the preparation of nitrones which react preferentially with hydroxyl radical over superoxide.4 Spin trapping12 has proven to be a powerful tool for the identification of biologically generated transient One difficulty encountered in the spin trapping of certain free radical intermediates, for example superoxide, is the rather slow rate (-10 M-hec-1) with which nitrones react with the intermediate. Thus potentially toxic concentrations of the nitrone are required for efficient trapping in biological systems. Our aim in this section is to develop nitrone-based spin traps with improved sensitivity owing to isotopic substitution. Our approach is based on the pioneering work of Beth et al.5 and Janzen et a1.6 These groups showed that a significant increase in ESR sensitivity could be achieved by preparing nitroxides that contain 2H and 15N in place of IH and 14N, respectively. Herein we report the synthesis of a family of such isotopically substituted nitrones and a study of their ability to spin trap superoxide and hydroxyl radicals generated from a model superoxide-generating system. 00GcX; C I I RyJ RQ; /O' 4, X, Y = "N / O H 1, X,Y=15N D 7, R=C&;N=% 8, R = CH3; N 14N 9, R = CD3; N = 15N 10, R = CH3; N = 14N

This volume is intended as a companion to the study of Cicero's oratory and rhetoric for both students and experts in the field: for the neophyte, it provides a starting point; for the veteran Ciceronian scholar, a place for renewing the dialogue about issues concerning Ciceronian oratory and rhetoric; for all, a site of engagement at various levels with Ciceronian scholarship and bibliography. The book is arranged along roughly chronological lines and covers most aspects of Cicero's oratory and rhetoric. The particular strength of this companion resides in the individual, often very original approach to sundry topics by an array of impressive contributors, all of whom have spent large portions of their careers concentrating upon the oratorical and rhetorical oeuvre of Cicero. A bibliography of relevant items from the past 25 years, keyed to specific Ciceronian works, completes the volume. Brill's Companion to Cicero will become the standard reference work on Cicero for many years.

Inhibition of hexose transport by N-ethylmaleimide was studied with regard to alkylation of different types of sulfhydryl group on the hexose carrier of the human erythrocyte. Uptake of 3-O-methylglucose was progressively and irreversibly inhibited by N-ethylmaleimide, with a half-maximal effect at 10–13 mM. A sulfhydryl group known to exist on the exofacial carrier was not involved in transport inhibition by N-ethylmaleimide, since reversible protection of this group by the impermeant sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) had no effect on the ability of N-ethylmaleimide to inhibit transport, or on its ability to decrease the affinity of the exofacial carrier for maltose. Nevertheless, the exofacial sulfhydryl was quite reactive with N-ethylmaleimide, since it was possible using a differential labeling technique to specifically label this group in protein-depleted ghosts with a half-maximal effect at 0.3 mM N-[ 3 H ] ethylmaleimide , and to localize it to the Mr 19 000 tryptic carrier fragment. Transport inhibition by N-ethylmaleimide correlated best with labeling of a single cytochalasin B-sensitive internal sulfhydryl group on the glycosylated Mr 23 000– 40 000 tryptic fragment of the carrier, which was half-maximally labeled at about 4 mM reagent. Whereas N-ethylmaleimide readily alkylates the exofacial carrier sulfhydryl, it inhibits transport by reacting with at least one internal carrier sulfhydryl located on the glycosylated tryptic carrier fragment.

Diabetic subjects have previously been found to have increased secretion of immunoreactive gastric inhibitory polypeptide (IR-GIP) in response to oral glucose or triglyceride compared with normal subjects. In the present study, a group of 19 diabetics was selected with milder glucose intolerance (fasting plasma glucose = 109 mg. per deciliter) than those reported previously. In these diabetics no enhancement of IRGIP responses was found to either 75 gm. oral glucose or 50 ml. oral corn oil when compared with 13 subjects with normal carbohydrate tolerance. Some of the subjects were obese and, when assessed independently from glucose tolerance, obesity was associated with higher integrated IRGIP responses to oral glucose but not to oral corn oil. The degree of obesity (92 to 154 per cent ideal body weight) did not correlate with fasting or stimulated IRGIP responses of the 32 subjects. One hour after ingestion of 50 ml. corn oil, when IRGIP concentrations were maximal but glucose and insulin remained unaffected, the diabetic subjects showed increased relative acute-phase insulin secretion to a 20 gm. pulse of intravenous glucose compared with that from a glucose pulse alone (p < 0.01). Subjects with normal carbohydrate tolerance had no increase in relative acute insulin response to glucose after triglyceride priming. The relative second-phase insulin response (10 to 30 min.) increased significantly with triglyceride priming in both diabetics (p < 0.01) and nondiabetics (p < 0.05). Although glucose disappearance rates (Kg) correlated with relative insulin responses, the increase in insulin secretion with triglyceride priming was not associated with an increase in Kg. Although obese subjects show increased stimulated GIP responses to oral glucose, patients with mild diabetes have normal GIP secretion to oral glucose and to triglyceride, but show a GIP-associated increase in both phases of glucose-induced insulin secretion.

The effects of ATP on glucose transport and metabolism were studied in rat adipocytes. Over a concentration range of 10–250 μ m , ATP was found to inhibit several aspects of adipocyte glucose metabolism, particularly when stimulated by insulin. Much of the effect of ATP on glucose metabolism appeared related to impairment of glucose transport, reflected by inhibition of both basal and insulin-stimulated rates of 3- O -methylglucose transport. ATP inhibited the V of insulin-stimulated 3- O -methylglucose transport, but had no effect on the K m . The inhibitory effects of ATP were much less apparent when cells were preincubated with insulin, suggesting that ATP inhibited only the components of hexose transport not yet activated by the hormone. At very high medium glucose concentrations, where transport was no longer rate limiting for metabolism, there was no inhibition of glucose oxidation by 250 μ m ATP. However, when hexose transport was blocked with cytochalasin B (50 μ m ), a small inhibitory effect of ATP persisted on basal and insulin-stimulated glucose and fructose oxidation, suggesting that intracellular metabolism was impaired. The mechanism of the intracellular effect did not appear to be caused by uptake of exogenous ATP. These studies provide further evidence that energy metabolism may play an important role in the regulation of facilitated glucose transport.

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.

The plasma concentrations of gastric inhibitory polypeptide (IR-GIP), insulin, and glucose were measured in 5 dogs after administration of various constituents of food. IR-GIP responses produced by galactose and fructose were minimal compared to those of glucose. Corn starch caused a significant early increase in insulin and glucose concentrations, although the rise in IR-GIP was delayed. The responses after starch were delayed, possibly due to the necessity for hydrolytic cleavage. Neither individual fatty acids (palmitic, oleic, and linoleic), monoolein, nor glycerol produced increases in IR-GIP comparable to those observed with lesser amounts (on a molar basis) of corn oil alone. None of four amino acids tested orally (arginine, leucine, alanine, and phenylalanine) caused increases in IR-GIP concentrations, but they increased insulin concentrations. Although glucose-induced IR-GIP responses were not inhibited by the amino acids (1 g/kg) tested, increasing concentrations (up to 1 g/kg) of either alanine or arginine caused progressive inhibition of corn-oil-stimulated IR-GIP responses, in spite of the expected amino-acid-induced rises in insulin concentrations. These results confirm the specificity of the IR-GIP secretory response for glucose and triglyceride, and suggest that certain food constituents (such as amino acids) may modify the GIP secretory response to a meal.

Many different models have been proposed to explain the complex binding behavior of insulin to its receptor, but a systematic comparison of models with experimental results is lacking. We have used network thermodynamic computer simulations to compare models of insulin binding against the results of several experimental tests designed to differentiate between the models. Six models of insulin binding were tested (simple, diffusion-reaction, conversion, dissociation, heterogeneous site, and two-step intramembrane) against results reported in the literature for isolated rat adipocytes. Although still a matter of experimental controversy, the criteria selected for modeling were curvilinear Scatchard plots, bi-or multi-exponential dissociation, insulin-accelerated dissociation, lack of dependence of the overall dissociation constant on receptor number, and receptor reserve. Using a given set of parameter values most appropriate for each model, none was able to account for all of the observed experimental results. This indicates both the complexity of the binding reaction and the need for further model development. The approach of using computer simulations to systematically test models against experimental results affords not only insight into the critical features of a model enabling it to pass a test, but also indicates potential experiments which might differentiate between models.

In one of his letters to Lucilius (Book 6,Ep.56), Seneca discusses the effects of noise and silence on study and contemplation.In the opening sections of the letter, he reveals that his current lodging is located above a bathhouse whence issue continually all sorts of irritating sounds. Seneca insists that such noises, despite their persistence, present no real distraction to one who possesses inner peace and a clear, untroubled mind (animum enim cogo sibi intentum esse nee avocari ad externa: omnia licet foris resonent, dum intus nihil tumultus sit,§5) and whose thoughts are ‘good, steadfast, and sure’ (nullus hominum aviumque concentus interrumpet cogitationes bonas solidasque iam et certas,§11).

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero-trans uptake of 3-O-methylglucose half-maximally at 5 mM, while more hydrophobic adenosine deaminase-resistant derivatives were ten- to 20-fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6-(L-2-phenylisopropyl)adenosine at 5 degrees C in a dose-dependent fashion (EC50 = 240 microM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6-(L-2-phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 microM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythrocyte ghosts. The cross-reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.

S-(Bismaleimidomethyl ether)cysteine (Cys-Mal) was synthesized as a probe for reactive thiol groups on the erythrocyte glucose carrier. Although Cys-Mal entered cells, its reaction with intracellular GSH prevented alkylation of endofacial membrane proteins, limiting its effect to the cell surface at concentrations below 5 mM. Cys-Mal irreversibly inhibited hexose transport half-maximally at 1.5 mM by decreasing the maximal rate of transport, with no effect on the affinity of substrate for the carrier. Reaction occurred with the outward-facing form of the carrier, but did not affect the ability of the carrier to change orientation. In intact cells, several exofacial proteins were labelled by [35S]Cys-Mal, including the band-4.5 glucose carrier, the labelling of which occurred on a single site sensitive to transport inhibitors. The reactive exofacial group was a thiol group, since both transport inhibition and band-4.5 labelling by Cys-Mal were abolished by the thiol-specific and impermeant compound 5,5′-dithiobis(2-nitrobenzoic acid). Selectivity for carrier labelling in cells was increased by a double differential procedure, which in turn allowed localization of the exofacial thiol group to the Mr 18,000-20,000 membrane-bound tryptic carrier fragment. In protein-depleted ghosts the exofacial thiol group was preferentially labelled at low concentrations of [35S]Cys-Mal, whereas with the reagent at 10 mM the Mr 26,000-45,000 tryptic carrier fragment was also labelled. Cys-Mal should be useful in the study of carrier thiol-group location and function.

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 m m ) had higher GSH contents (3.2 μg/10 6 cells) than in the absence of glucose (2.3 μg/10 6 cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H 2 O 2 to a concentration of 60 μ m to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 m m glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N -ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 m m GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 m m NADP + was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP + ] ratios may play a significant role in short-term control of the pentose phosphate cycle.

A nitrophenyl azide derivative of maltose, N -(4-azido-2-nitrophenyl)-maltosylamine (NAP-maltosylamine), was synthesized as a potential photoaffinity label for the hexose carrier of the rat adipocyte. This derivative inhibited 3- O -methylglucose uptake with a K i of 1.3 mM in the dark, while that of maltose was 10.0 mM. Carbon-14-labeled maltose and NAP-maltosylamine entered adipocytes via the hexose carrier, the latter in a concentrative fashion. Photolysis of NAP-[ 14 C]maltosylamine in the presence of an adipocyte low density microsomal membrane fraction labeled several electrophoretic bands. Among these are a 45 kDa band which showed features expected of the hexose carrier: its labeling was decreased 40% by d - but not l -glucose and pretreatment of intact adipocytes with insulin decreased labeling of the 45 kDa band by 10–40%, as predicted by the translocation theory of insulin-stimulated transport activation. These studies show the suitability of using carbon-1-modified sugar photoaffinity labels as probes for the hexose carrier and possibly of its regulation in rat adipocytes.

Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 μg/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.

The sulfhydryl reagent 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1 mM DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10 mM cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120 mM accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 microM cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfhydryl, but reaction of this group may not always "lock" the carrier in an outward-facing conformation.

The effect of glucose on lipolytic regulation was studied in isolated human adipocytes. Glucose enhanced adipocyte glycerol release in the presence and absence of the β-adrenergic agent ritodrine by 150–200% of control rates. The glucose effect was maximal at just >1 mM glucose and could not be attributed to prevention of a time-dependent decline in lipolysis. Glucose not only increased lipolytic stimulation at each of several concentrations of ritodrine but also enhanced the sensitivity to stimulation at low concentrations of the agent. Ritodrine-stimulated lipolysis was inhibited by insulin by 50–60%; although glucose increased absolute rates of lipolysis, it did not affect the relative inhibition of lipolysis by insulin or the sensitivity to the hormone. In investigating a possible cause of the glucose effect on lipolysis, it was found that the addition of adenosine deaminase increased lipolytic rates in the absence of glucose and blunted the relative stimulation of lipolysis by glucose, the latter implicating extracellular adenosine in the mechanism of the glucose effect.

Maltose-maleimide was synthesized as a potential affinity label for the facilitative hexose carrier with selectivity for exofacial sulphydryl groups. This reagent, although probably a mixture of isomers, did not significantly penetrate the plasma membrane of human erythrocytes at concentrations below 5 mM at 37 degrees C. When allowed to react to completion, it irreversibly inhibited the uptake of 3-O-methylglucose, with a half-maximal response at about 1.5-2.0 mM-reagent. The rate of transport inactivation was a saturable function of the maltose-maleimide concentration. Studies of reaction kinetics and effects of known transport inhibitors demonstrated that irreversible reaction occurred on the exofacial outward-facing carrier, although not at a site involved in substrate binding. Reaction of intact erythrocytes with [14C]maltose-maleimide resulted in labelling of a broad band 4.5 protein of Mr (average) 45,000-66,000 in electrophoretic gels. This protein was very likely the hexose carrier, since its labelling was inhibited by cytochalasin B. Exofacial band 4.5 labelling was stoichiometric with respect to transport inhibition, yielding an estimated 300,000 carriers/cell. These results suggest that the exofacial sulphydryl which reacts with maltose-maleimide is distinct from the substrate binding site on the hexose carrier, but that it confers substantial labelling selectivity to impermeant maleimides. Additionally, the high efficiency of carrier labelling obtained with maltose-maleimide is useful in quantifying numbers of carriers in whole cells.

Androgenic steroids, which are potent inhibitors of facilitated hexose transport in human erythrocytes, were tested as possible natural photolabels of the hexose carrier protein. Androstenedione, which inhibited 3-O-methylglucose uptake half-maximally at 30-50 microM (EC50), was the most potent inhibitor of the photolabile steroids tested. It appeared to interact directly with the carrier, since it (1) inhibited equilibrium [3H]cytochalasin B binding to high affinity D-glucose-sensitive sites in both intact cells (EC50 = 63 microM) and protein-depleted ghosts (EC50 = 61 microM), (2) inhibited cytochalasin B photolabeling of the band 4.5 carrier region in electrophoretic gels of protein-depleted ghosts (EC50 = 50 microM), and (3) underwent photoincorporation into the same gel region in a D-glucose- and cytochalasin B-sensitive fashion. However, Dixon plots for inhibition of both cytochalasin B binding and transport were upward-curving, indicating the binding of more than one molecule of androstenedione to the carrier. The photoincorporation of androstenedione into band 4.5 protein was both time- and concentration-dependent, and not associated with damage to unlabeled carrier. It probably occurred by activation of the alpha, beta-unsaturated ketone on the steroid rather than indirectly by photoactivation of a group on the carrier protein, as occurs with cytochalasin B. Although androstenedione may bind to more than one region of the carrier, as well as to other non-carrier proteins, tryptic digestion of photolabeled ghosts produced a labeled Mr = 18,000-20,000 fragment, the labeling of which was inhibited by cytochalasin B, and which had an electrophoretic mobility similar to the major labeled tryptic fragment in cytochalasin B-labeled ghosts. These data suggest that androstenedione interacts directly with the hexose carrier and that it or other similar naturally photolabile steroids may serve as useful probes for structural dissection of the carrier protein.

Sulfhydryl-reactive derivatives of glucosamine were synthesized as potentially transportable affinity labels of the human erythrocyte hexose carrier. N-Maleoylglycyl derivatives of either 6- or 2-amino-2-deoxy-D-glucopyranose were the most potent inhibitors of 3-O-methylglucose uptake, with concentrations of half-maximal irreversible inhibition of about 1 mM. Surprisingly, these derivatives were very poorly transported into erythrocytes. They reacted rather with an exofacial sulfhydryl on the carrier following a reversible binding step, the latter possibly to the exofacial substrate binding site. However, their reactivity was determined primarily by access to the exofacial sulfhydryl, which, as predicted by the one-site model of transport, required a carrier conformation with the exofacial substrate binding site exposed. Once reacted, the carrier was "locked" in a conformation unable to reorient inwardly and bind cytochalasin B. In intact erythrocytes the N-maleoylglycyl derivative of 2-[3H]glucosamine labeled predominantly an Mr 45,000-66,000 protein on gel electrophoresis in a quantitative and cytochalasin B inhibitable fashion. By use of changes in carrier conformation induced by competitive transport inhibitors in a "double" differential labeling method, virtually complete selectivity of labeling of the carrier protein was achieved, the latter permitting localization of the reactive exofacial sulfhydryl to an Mr 18,000-20,000 tryptic fragment of the carrier.

The cardiovascular effects of vitamin C (VitC) could be mediated by epoxyeicosatrienoic acids (EETs). We aimed to study the mechanism of VitC-dependent microsomal formation of cis- and trans-EETs and the regulation of EET levels in rat isolated perfused kidneys and in vivo. VitC biphasically stimulated rat kidney microsomal cis- and trans-EET formation in a ratio of 1:2, involving the participation of lipid hydroperoxides (LOOHs), Fe2+ , and cytochrome P450 (CYP). Levels of LOOHs correlated with microsomal EET production. LOOH stimulation of CYP isoforms resulted in preferred trans- over cis-EET formation from arachidonic acid and was associated with the cleavage of LOOHs, which indicated a CYP peroxy-genase activity. EETs contributed to VitC-induced vasodilator responses in rat isolated perfused kidneys. VitC (1 mg/ml) given in the drinking water for 9 days doubled rat urinary EET excretion, increased plasma levels of EETs, mostly trans-EETs, by 40%, and reduced plasma levels of 20-hydroxyeicosatetraenoic acid. Depletion of VitC in brain cortex and kidney tissues by more than 20- and 50-fold, respectively, in gulonolactone oxidase-knockout mice was associated with mild increases in tissue EETs. These data suggest that LOOHs are a determinant factor for EET formation in vivo in which VitC exerts a key regulatory effect. VitC-activated CYP peroxygenase activity may represent a CYP interaction with lipoxygenases and cyclooxygenases to mediate the cardiovascular effects of VitC via formation of EETs.