Endothelium-Derived Hyperpolarizing Factor, But Not Nitric Oxide, Is Reversibly Inhibited by Brefeldin A
Abstract The subcellular localization of the enzymes synthesizing endothelium-derived vasodilator autacoids has been proposed to play a role in determining the ability of endothelial cells to enhance autacoid production in response to stimulation. We therefore investigated the effects of brefeldin A–induced disruption of the Golgi apparatus and Golgi-plasma membrane trafficking on the production of nitric oxide (NO), prostacyclin, and the endothelium-derived hyperpolarizing factor (EDHF) by native and cultured endothelial cells. In porcine coronary artery segments, brefeldin A (35 μmol/L, 90 minutes) did not affect relaxations to sodium nitroprusside or the K+ channel opener cromakalim but elicited a rightward shift in the concentration-response curve to bradykinin without altering the maximum vasodilator response (Rmax). Brefeldin A failed to attenuate the bradykinin-induced, NO-mediated relaxation under depolarizing conditions but inhibited the bradykinin response under conditions of combined cyclooxygenase/NO synthase blockade, suggesting that this agent selectively interferes with the production of EDHF. Indeed, incubation of porcine coronary arteries with brefeldin A, which did not affect the bradykinin-induced accumulation of either cyclic GMP or 6-keto-prostaglandin F1α, markedly and reversibly attenuated the EDHF-mediated hyperpolarization of detector smooth muscle cells in a patch-clamp bioassay system. The microtubule destabilizer nocodazole also affected both the EC50 and Rmax to bradykinin in porcine coronary arteries. Since EDHF is thought to be a cytochrome P450–derived metabolite of arachidonic acid and both brefeldin A and nocodazole are known to interfere with the targeting of cytochrome P450 from the Golgi apparatus to the plasma membrane, it is conceivable that brefeldin A inhibits EDHF formation by preventing the targeting of the EDHF-synthesizing enzymes to the plasma membrane.
- endothelium-derived hyperpolarizing factor
- nitric oxide
- cytochrome P450
- brefeldin A
- endothelial cells
Endothelial cells synthesize and release the vasodilator autacoids NO and prostacyclin as well as the so-called EDHF (for review, see Reference 11 ). While the intracellular site of synthesis of the more unstable autacoids (NO and EDHF) is likely to have important implications on their sphere of influence and thus biological activity, the cellular distribution of the respective generating enzymes and the relationship between localization and enzymatic activity are unclear.
There are conflicting reports concerning the major intracellular site of NO formation and the impact of endothelial NO synthase (NOS III) localization on the generation of bioactive NO. Histochemical studies suggested that NOS III is predominantly situated in the Golgi apparatus,2 3 and association with this organelle is reportedly necessary for the enzyme to respond to intracellular signals and for efficient NO synthesis.4 However, more recent studies have indicated that functionally active NOS III is concentrated in caveolin-rich membrane domains,5 6 although NOS III, and the caveola marker protein caveolin,7 may cycle from the Golgi apparatus to caveolae.
Compelling evidence exists for the release of a diffusible endothelium-derived autacoid that induces relaxation by hyperpolarizing the underlying smooth muscle cell layers.8 9 10 However, since the chemical identity of this EDHF is still elusive, no information is available regarding the intracellular localization of the “EDHF synthase.” Pharmacological studies have, however, suggested that the EDHF generated by the coronary10 11 12 13 14 and femoral endothelium15 may be an arachidonic acid metabolite generated by cytochrome P450–dependent enzymes, which are normally present in the endoplasmic reticulum.16
The aim of the present study was to determine the role of the Golgi apparatus and the endoplasmic reticulum in the synthesis of all three vasodilator autacoids, ie, NO, prostacyclin, and EDHF, by endothelial cells. We therefore investigated the effects of brefeldin A, which causes reversible disassembly of the Golgi apparatus and swelling of the endoplasmic reticulum,17 on autacoid formation in native porcine coronary artery and cultured human endothelial cells. Since brefeldin A also affects microtubule-dependent transport processes, we further studied the effect of interfering with microtubule polymerization and depolymerization on endothelial autacoid production.
Bradykinin was from Bachem Biochemica GmbH; HEPES and L-NNA were from Serva. Taxol was obtained from Calbiochem, and nocodazol from Fluka Chemika. Diclofenac (Voltaren injection solution) was from CIBA-Geigy, and U46619 (9,11-dideoxy-11α,9 α-epoxymethano-prostaglandin F2α) was provided by Upjohn. The specific NOS III antibody was purchased from Transduction Laboratories. Brefeldin A, cromakalim, phenylephrine, ACh, isobutyl-methyl-xanthine (IBMX), SNP, and all other chemicals were purchased from Sigma Chemical.
Human umbilical vein or porcine aortic endothelial cells were seeded on fibronectin-coated culture dishes containing medium M-119 (GIBCO Berlin) and 20% heat-inactivated fetal calf serum (Vitromex) supplemented with penicillin (50 U/mL), streptomycin (50 μg/mL), l-glutamine (1 mmol/L), glutathione (5 mg/mL), and l(+)-ascorbic acid (5 mg/mL). Rat aortic smooth muscle cells were isolated and cultured as described.10 Confluent cultures of smooth muscle cells were passaged by using trypsin-EDTA (1 mmol/L), and experiments were performed using cells between passages 12 and 16.
Organ Bath Studies
Porcine coronary artery or rabbit aortic rings (3 mm in length) were mounted between force transducers (Scaime) and a rigid support for measurement of isometric force and incubated in organ baths containing warmed (37°C), oxygenated (95% O2, 5% CO2) Krebs-Henseleit solution (pH 7.4) of the following composition (mmol/L): NaCl 118.4; NaHCO3 25.0; KCl 4.7; CaCl2 1.6; KH2PO4 1.2; MgSO4 1.2; and glucose 11.1.
Porcine Coronary Arteries
Passive tension was adjusted over a 60-minute equilibration period to 5 g; thereafter, the segments were repeatedly exposed to KCl (70 mmol/L) until stable constrictions were obtained. After washing, 10 g of tension (≈70% of the maximal constriction) was applied by constriction using U46619 (0.1 to 0.3 μmol/L). The integrity of the endothelium was assessed using the endothelium-dependent vasodilator bradykinin (1 μmol/L), and vessels exhibiting <70% relaxation were discarded.
Passive tension was adjusted over a 30-minute equilibration period to 2 g; thereafter, the segments were repeatedly exposed to phenylephrine (1 μmol/L) until stable constrictions were obtained (≈70% of the maximal constriction). The integrity of the endothelium was assessed using ACh (1 μmol/L), and vessels exhibiting <70% relaxation were discarded.
All vessels used were preconstricted to the same tension using the appropriate agonist before the addition of the vasodilator (bradykinin or ACh). As the relaxant responses observed were transient, the dose-response curve represents the peak response observed to cumulative applications of the agonists.
Detection of EDHF Release
The release of EDHF was detected by recording changes in the membrane potential of cultured rat aortic smooth muscle cells exposed to the effluate of a perfused porcine coronary artery. Briefly, a segment of porcine coronary artery was cannulated at both ends, mounted in an organ chamber, and perfused intraluminally (1 mL/min) with PSS of the following composition (mmol/L): NaCl 140; KCl 4.7; MgCl2 1; CaCl2 1.3; glucose 5; HEPES 10 (pH 7.4, 37°C) containing the NOS inhibitor L-NNA (100 μmol/L) and the cyclooxygenase inhibitor diclofenac (1 μmol/L). Effluate from the segment superfused cultured smooth muscle cells, the membrane potential of which was recorded by the slow whole-cell configuration of the patch-clamp technique using nystatin (100 μg/mL) in the pipette as described.10 18 The cell membrane potentials, measured in the current clamp mode, were recorded continuously. Only detector smooth muscle cells that had a stable resting membrane potential for more than 2 minutes and exhibited no further change in the input resistance were used in the bioassay system.
Detection of Prostacyclin Release
In a separate series of experiments, the concentration of 6-keto PGF1α, the stable hydrolysis product of prostacyclin, was measured by a specific radioimmunoassay either in the effluate of perfused (0.5 mL/min) porcine coronary arteries (collected for 3 minutes before and after application of bradykinin, 0.1 μmol/L) or in the supernatant of cultured human endothelial cells under resting conditions and after stimulation with bradykinin (10 nmol/L, 5 minutes).
Determination of Cyclic GMP Concentration
The concentration of cyclic GMP in confluent cultured human endothelial cells under resting conditions and after stimulation with bradykinin (10 nmol/L, 5 minutes) in the presence of the phosphodiesterase inhibitor IBMX (0.1 mmol/L) was determined with a specific radioimmunoassay.
Isolation of Caveolin-Rich Membrane Domains and Determination of NOS III Activity
Freshly isolated porcine aortae were slit longitudinally, mounted in an open chamber, washed twice in HEPES-modified Tyrode solution (mmol/L: NaCl 132, KCl 4, CaCl2 1, MgCl2 0.5, HEPES 9.5, and glucose 5), and the exposed endothelial layer was incubated at 37°C in the presence and absence of agonists as indicated in “Results.” Thereafter, the incubation was stopped by exchanging the incubation medium with ice-cold HEPES buffer, and the cells were harvested by scraping. Thereafter, the cytosolic cell fraction and caveolin-rich membrane domains were prepared by detergent-free sequential centrifugation as described,19 and the presence of NOS III in each fraction was determined by Western blotting after separation of proteins by SDS-PAGE. NOS activity in aliquots (4 μg protein) of the cytosolic and caveolae cell fractions was determined by assessing the L-NNA (1 mmol/L)–sensitive conversion of l-[3H]arginine to l-[3H]citrulline as described previously.20
Assay of Cytochrome P450 Activity
Cytochrome P450–dependent metabolic activity was assayed as the dealkylation of 7-ethoxyresorufin in confluent porcine aortic endothelial cells cultivated in the absence and presence of β-naphthoflavone (3 μmol/L, 48 hours).10
Samples (cells or vessels) were fixed using 4% formaldehyde in phosphate-buffered saline for 30 minutes, permeabilized in 0.05% Triton X-100 for 10 minutes followed by blocking in 100 mmol/L glycine for 10 minutes. Thereafter, samples were incubated in a reaction buffer consisting of 0.01% Triton X-100, 0.1% carboxylated bovine serum albumin (AURION, Netherlands) in phosphate-buffered saline, pH 7.6. The fixed samples were incubated for 1 hour with a mouse monoclonal antibody directed against the NOS III (3 μg/mL, Transduction Laboratories) in a 50-mmol/L TRIS solution (pH 7.5) containing NaCl 200 mmol/L, Triton X-100 0.2%, bovine serum albumin 3%, and horse serum 10%. Samples were then incubated with a biotinylated donkey anti-mouse antibody for 1 hour followed by streptavidin-Cy2 (Biotrend). After nuclear staining with 7-amino-actinomycin D (Molecular Probes), the preparations were mounted in Mowiol (Hoechst) and examined using a confocal microscope (Leica TCS).
Relaxant responses are given as percentage relaxation relative to preconstriction levels. All data in the figures and in the text are expressed as mean±SEM of n experiments using segments from different animals. Statistical analysis was performed by one-way ANOVA followed by a Bonferroni t test or by the Mann-Whitney test for unpaired data where appropriate, with values of P<.05 being considered statistically significant.
Effects of Brefeldin A on Endothelium-Dependent and -Independent Relaxations
In porcine coronary artery segments preconstricted with U46619 (0.1 to 0.3 μmol/L), brefeldin A (35 μmol/L, 90 minutes) elicited a rightward shift in the concentration-response curve to bradykinin (Fig 1⇓, Table 1⇓) but did not alter the maximum vasodilator response (Rmax). The effect of brefeldin A was selective for endothelium-dependent vasodilation because brefeldin A treatment did not affect relaxations induced by SNP (Table 1⇓) or the K+ channel opener cromakalim (0.1, 1 μmol/L; Rmax, control 89±9% versus 98±2% after brefeldin A treatment).
Effect of Brefeldin A on NO Production
In porcine coronary artery segments preconstricted with depolarizing concentrations of KCl (30 mmol/L), brefeldin A failed to affect bradykinin-induced relaxations (Fig 2A⇓), which under these conditions are solely mediated by NO as they were abolished in the presence of L-NNA (100 μmol/L; data not shown). Similarly, in phenylephrine-preconstricted rabbit aortic rings, which we have previously demonstrated produce little or no EDHF,21 ACh (1 nmol/L to 30 μmol/L) elicited a concentration-dependent, NO-mediated relaxation. This response was insensitive to brefeldin A (35 μmol/L, 90 minutes) but was abrogated by L-NNA (100 μmol/L; data not shown).
In cultured human endothelial cells, the L-NNA–sensitive increase in cyclic GMP levels was taken as an index of NO production. Pretreatment of these cells with brefeldin A (35 μmol/L) affected neither basal cyclic GMP levels (control, 14.2±1.2; brefeldin A, 12.0±0.5 pmol cGMP/mg protein) nor the bradykinin (10 nmol/L)– induced increase in cyclic GMP production (control, 231±18; brefeldin A, 260±28 pmol cGMP/mg protein, n=8). NOS III activity was also determined in isolated caveolin-rich membrane domains and in the cytosolic fractions enriched in the Golgi marker enzyme α-mannosidase II, which were prepared from native porcine endothelial cells after treatment with either solvent or brefeldin A (35 μmol/L, 90 minutes). Endothelial NO production as assessed by the formation of l-citrulline from l-arginine was enhanced in the caveolae relative to the Golgi apparatus (Fig 2B⇑). This was consistent with the finding that the ratio of NOS III detected in the Golgi and caveolae was 1:5.6. Brefeldin A was without significant effect on the total NOS activity but altered the distribution of NOS activity (Fig 2B⇑) and protein between the caveolae fraction and the Golgi apparatus (ratio 1:3.4).
Experiments using indirect immunofluorescence coupled to confocal microscopy demonstrated that in confluent cultured endothelial cells, NOS III is concentrated at the cell periphery and in a perinuclear site, identified as the Golgi apparatus (Fig 3A⇓). After incubation of endothelial cells with brefeldin A (35 μmol/L), the intense perinuclear signal was no longer observed, suggestive of a marked disorganization of the Golgi apparatus (Fig 3B⇓). Identical results were obtained with native porcine coronary endothelial cells.
Effect of Brefeldin A on Prostacyclin Release
In porcine coronary arteries, prostacyclin does not contribute appreciably to agonist-induced endothelium-dependent relaxations as confirmed by the lack of effect of diclofenac on bradykinin-induced relaxations (data not shown). Therefore, formation of prostacyclin was assessed as the accumulation of the stable derivative 6-keto-PGF1α in the effluate from porcine coronary artery segments. The application of bradykinin (0.1 μmol/L) elicited an increase in the release of 6-keto-PGF1α from 1437±190 to 2479±296 pg 6-keto-PGF1α/mL (n=8). Pretreatment with brefeldin A (35 μmol/L) affected neither the basal (1564±203 pg 6-keto-PGF1α/mL) nor the bradykinin-induced increase in 6-keto-PGF1α (2291±278 pg 6-keto-PGF1α/mL). Brefeldin A also failed to affect the bradykinin-induced increase in 6-keto-PGF1α in cultured human endothelial cells (control, 72.5±17.5 versus 1925±213 pg 6-keto-PGF1α/mg protein; brefeldin A, 87.5±25.0 versus 2275±174 pg 6-keto-PGF1α/mg protein, in the absence and presence of bradykinin, respectively).
Inhibition of EDHF Release by Brefeldin A
In the combined presence of diclofenac (1 μmol/L) and L-NNA (100 μmol/L), conditions under which endothelium-dependent relaxation is mediated entirely by EDHF, brefeldin A induced a pronounced rightward shift in the concentration-response curve to bradykinin as well as a marked attenuation of Rmax in porcine coronary arteries. The inhibitory effect of brefeldin A on EDHF production was reversible, since washout (3 hours) resulted in an almost complete restoration of the bradykinin-induced EDHF-mediated relaxation (Fig 4C⇓).
The effects of brefeldin A on EDHF production were further analyzed in a patch-clamp setup for the detection of EDHF. In this system, the effects of brefeldin A on EDHF release from the donor endothelium were investigated separately from effects on the membrane potential and EDHF-induced hyperpolarization of the detector cells.
Stimulation of the donor porcine coronary arteries with bradykinin (0.1 μmol/L) in the presence of both diclofenac and L-NNA led to the hyperpolarization (14±2 mV, n=6) of detector smooth muscle cells situated downstream from the donor. After pretreatment of the donor segments with brefeldin A (35 μmol/L), the bradykinin-induced, EDHF-mediated hyperpolarization of detector smooth muscle cells was significantly reduced (Fig 5⇓). Removal of brefeldin A from the donor perfusate (3 hours) resulted in a substantial restoration of the smooth muscle cell hyperpolarization elicited by effluate from bradykinin-stimulated arterial segments (Fig 5⇓). The effect of brefeldin A could be attributed to inhibition of the release of EDHF from the donor artery because the direct application of brefeldin A (35 μmol/L, 90 minutes) to smooth muscle cells failed to affect either the resting membrane potential or the hyperpolarization elicited by the perfusate from bradykinin-stimulated donor arteries (data not shown).
Brefeldin A (35 μmol/L) was without effect on basal P450 oxygenase activity as assessed in either solvent or β-naphthoflavone-treated porcine aortic endothelial cells (control, 4.5±0.5 versus brefeldin A; 4.7±0.4 nmol resorufin · L−1 · min−1 in solvent-treated cells and control, 68±8 versus brefeldin A, 71±7 nmol resorufin · L−1 · min−1 in β-naphthoflavone–treated cells; n=12).
Effects of Nocodazol and Taxol on Agonist-Induced Relaxations in Porcine Coronary Arteries
The effects of interfering with microtubule polymerization and depolymerization on endothelial autacoid production were investigated using nocodazole and taxol,22 23 which have been previously shown to interfere with microtubule organization.24 Treatment of porcine coronary arteries with nocodazole (30 μmol/L, 90 minutes) failed to affect the mainly NO-mediated relaxation of arteries in response to bradykinin (Table 2⇓). A distinct attenuation of both the EC50 and Rmax to bradykinin was observed when the effects of nocodazole were investigated in rings pretreated with L-NNA; however, this effect just failed to attain statistical significance. Endothelium-independent relaxation to SNP was not affected by nocodazole (Table 2⇓). Taxol (10 μmol/L, 120 minutes), which reportedly stabilizes microtubules, was without effect on the bradykinin-induced production of either NO or EDHF (Table 2⇓).
In the present study, we demonstrated that in both native and cultured endothelial cells the disruption of the Golgi apparatus by brefeldin A does not alter NO or prostacyclin formation but leads to a pronounced inhibition of EDHF production. Brefeldin A is therefore the first inhibitor described to date that selectively and reversibly interferes with the generation of EDHF in endothelial cells.
Two intracellular pools of NOS III protein, the Golgi apparatus and caveolae, have been described in native and cultured endothelial cells.5 6 However, contradictory reports exist regarding the relative contribution of these intracellular sites to the production of bioactive NO.3 4 5 Indeed, while functionally active NOS III is concentrated in caveolae prepared from native and cultured endothelial cells,5 6 brefeldin A–induced disruption of the Golgi apparatus has been reported to abrogate NO production in cerebrovascular endothelial cells.25 In the present study, brefeldin A failed to affect NO production in either native or cultured endothelial cells despite complete loss of the immunofluorescent perinuclear NOS III signal.
Biochemical analysis revealed that NOS III protein was up to six times more concentrated in caveolae than in the Golgi-containing cytosolic fraction, a finding that is reflected by the differences in NOS activity detected in the two subcellular fractions. Although brefeldin A did not attenuate overall NOS activity, it did induce a distinct decrease in caveolar and an increase in cytosolic NO formation. This brefeldin A–induced change in NOS activity was accompanied by a shift in the distribution of NOS III between these two cellular compartments. Although under depolarizing conditions brefeldin A did not attenuate NO-mediated relaxation, we cannot exclude the possibility that the shift in the concentration-response curve to bradykinin under control conditions may be partially attributable to the effects of brefeldin A on NOS III compartmentalization. Moreover, since brefeldin A inhibits endoplasmic reticulum/Golgi trafficking, our results suggest that there is a rapid turnover of the caveolae-associated NOS III and that a continual exchange of NOS III takes place between the Golgi apparatus and caveolae in native endothelial cells.
The lack of effect of brefeldin A on prostacyclin formation in porcine coronary and cultured human endothelial cells indicates that disorganization of the Golgi apparatus and endoplasmic reticulum has, as expected, no effect on the enzymatic activity of either cyclooxygenase I or phospholipase A2, both of which are cytosolic enzymes.
The reversible inhibition of the bradykinin-induced, NO/prostacyclin–independent vasodilation of porcine coronary arteries suggests that brefeldin A selectively affects the EDHF synthesis/signaling pathway. EDHF-induced hyperpolarization is brought about by an increase in the K+ conductance of vascular smooth muscle cells via activation of Ca2+-dependent K+ (K+Ca) channels and is abolished by selective K+Ca channel inhibitors.9 10 12 14 The transient nature of the EDHF-induced hyperpolarization observed in patch-clamp bioassay is also evident in intact vascular segments where the hyperpolarization of smooth muscle usually precedes and is more transient than the accompanying relaxation induced by endothelium-dependent vasodilators.26 27 28
Since neither the resting membrane potential nor the hyperpolarization induced by EDHF released from an untreated segment were affected by prolonged incubation of detector cells with brefeldin A, the inhibitory effect of brefeldin A on the EDHF response appears unrelated to effects on the EDHF-mediated activation of K+Ca channels. Moreover, in our patch-clamp setup, an attenuated EDHF-induced hyperpolarization of detector vascular smooth muscle cells was observed only after incubation of the donor artery with brefeldin A. Additional studies using a variety of different cell types have also failed to observe a direct inhibitory effect of brefeldin A on K+, Cl−1, and Ca2+ channel activity.29 30 31
It is possible only to speculate on the mechanism by which brefeldin A inhibits EDHF synthesis, since the chemical identity of the EDHF produced by the coronary endothelium has not been fully elucidated. Experimental evidence, however, suggests that this factor may be a cytochrome P450–derived metabolite of arachidonic acid, such as an epoxyeicosatrienoic acid.13 14 32 Moreover, induction of cytochrome P450 enzymes using β-naphthoflavone has been shown to enhance the release of EDHF from native rat mesenteric as well as cultured porcine and human endothelial cells.10 28 Recently, a specific cannabinoid receptor (CB1) antagonist has been shown to attenuate EDHF-induced relaxation of isolated rat mesenteric vessels, whereas anandamide, an endogenous cannabinoid, elicited “EDHF-like” relaxations.33 These results do not necessarily imply that two candidates for EDHF exist. Indeed, anandamide can be enzymatically synthesized from, and may well prove to be a carrier of, arachidonic acid,34 the putative precursor of EDHF. Moreover, anandamide can be metabolized by cytochrome P450, and the induction of P450 enzymes results in the increased formation of several anandamide metabolites.35 The mechanism by which brefeldin A attenuates EDHF formation, however, does not appear to be related to a decreased availability of arachidonic acid, since the lack of effect of brefeldin A on prostacyclin formation suggests that the liberation of arachidonic acid is not inhibited. Nor does brefeldin A treatment result in the global inhibition of cytochrome P450–dependent monooxygenase activity, since this agent did not affect the ability of endothelial cells to dealkylate the P450 substrate 7-ethoxyresorufin. Rather, the effects of brefeldin A on the trafficking of proteins between the endoplasmic reticulum, Golgi apparatus, and the plasma membrane may be responsible for the observed inhibition of EDHF release. Although cytochrome P450 is synthesized on polyribosomes bound to the endoplasmic reticulum and is cotranslationally inserted into the endoplasmic reticulum membrane,36 there is an extensive flow of vesicles to the Golgi apparatus as well as a microtubule-dependent transport from the Golgi to the plasma membrane.37 Indeed, the trafficking of P4502B between the Golgi apparatus and the plasma membrane has been demonstrated in cultured rat hepatocytes.38 In the latter study, moreover, incubation of hepatocytes with brefeldin A for up to 2 hours essentially suppressed the expression and activity of P4502B on the plasma membrane without inducing a measurable decrease in total cellular P450 activity.38 That a similar phenomenon is responsible for the effects of brefeldin A on EDHF production is supported by the observation that the microtubule inhibitor nocodazole also attenuated EDHF-mediated relaxations in the present study. The finding that the inhibitory effect of nocodazole was not as marked as that induced by brefeldin A may also be explained by the fact that this agent was only half as effective as brefeldin A in reducing the expression of P450 at the plasma membrane.38 Corroboration of this hypothesis requires experiments using antibodies raised against the cytochrome P450 implicated in the generation of EDHF and must therefore wait until a suitable candidate has been proposed from the multitude of isoforms described to date.39
In summary, our data indicate that acute disruption of the Golgi apparatus and inhibition of protein transport to the plasma membrane by brefeldin A have little effect on NO or prostacyclin production but almost completely inhibits the synthesis of EDHF by endothelial cells. Thus, brefeldin A, which inhibits a decisive step in the EDHF-forming pathway (probably by preventing the targeting of the necessary enzymatic machinery to the plasma membrane), will be an invaluable tool for the identification of this elusive factor.
This study was supported by the Deutsche Forschungsgemeinschaft (Bu 436/6-1) and the Commission of the European Communities (BMH4-CT96-0979). The authors are indebted to Isabel Winter, Andreas Schäfer and Michaela Stächele for expert technical assistance.
- Received March 3, 1997.
- Revision received March 19, 1997.
- Accepted June 6, 1997.
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