GH-RH antagonist (MZ-4-71) inhibits VEGF secretion and proliferation of murine endothelial cells

Angiogenesis plays a key role in solid tumor formation, invasiveness and metastasis. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is necessary in the process of neovascularisation. Antagonists of growth hormone-releasing hormone (GH-RH) have been shown to suppress both in vivo and in vitro growth and metastasis of many human cancer cell lines. The mechanisms that mediate the antitumorigenic actions of these antagonists involve direct and indirect pathways, but are not completely elucidated. We have examined the effect of GH-RH antagonist MZ-4-71 on proliferation activity and VEGF release from cultured murine endothelial cells HECa10 in vitro. MZ-4-71 at 10(-8) to 10(-6) M concentrations inhibited the proliferative activity of cultured cells and suppressed the release of VEGF into supernatants of 72 h endothelial cell cultures. To our knowledge this is the first study reporting antiangiogenic properties of GH-RH antagonists.     

In vivo and in vitro effects of cholecystokinin octapeptide on the release of growth hormone in rats

The effect of cholecystokinin octapeptide (CCK-8) on the release of growth hormone (GH) in rats was studied in vivo and in vitro. Intravenous injection of 5 micrograms/100 g BW of CCK-8 resulted in significant increase in the plasma GH level after 10 and 20 min. CCK-8 at concentrations of 10(-11)M to 10(-7)M also caused dose-dependent stimulation of GH release from dispersed cells of rat anterior pituitary. On the other hand, somatostatin (SRIF) inhibited GH release from dispersed cells of rat anterior pituitary in a dose-related manner at concentrations of 10(-7)M to 10(-9)M. Release of GH from the cells was increased by addition of K+ at high concentration (50 mM) in a Ca++-dependent manner. Addition of 10(-3)M verapamil to the incubation medium inhibited CCK-8-induced GH release from the cells. Addition of SRIF (10(-7)M) to the incubation medium inhibited GH release from the cells induced by CCK-8 or high K+ (50 mM). These results indicate that CCK-8 acts directly on the anterior pituitary cells to stimulate GH release and that calcium ion is involved in the mechanism of this effect.     

Leukotriene C4 stimulates angiogenesis in bovine carotid artery endothelial cells in vitro

In order to investigate the mechanism of angiogenesis involved in inflammatory processes, the effects of leukotrienes and prostaglandin E2 on in vitro tube formation of cultured vascular endothelial cells were examined. Endothelial cells from bovine carotid artery were cultured for 4 days between two layers of collagen gel and the lengths of organized tubes were quantitatively estimated with an image analyzer. Treatment with 10(-8)-10(-6)M of prostaglandin E2 increased the tubular lengths, and leukotriene C4 stimulated tube formation at far lower concentrations (10(-15)-10(-9)M) but leukotriene B4 and D4 were not effective on the tube formation. It was also found that endothelial cell migration was stimulated by almost the same concentrations of leukotriene C4 as those stimulating tube formation. These data suggest that leukotriene C4 is, at least, one of the important factors involved in angiogenesis during inflammatory processes.     

Vascular endothelial growth factor is modulated in vascular muscle cells by estradiol, tamoxifen, and hypoxia

Vascular endothelial growth factor (VEGF) promotes neovascularization, microvascular permeability, and endothelial proliferation. We described previously VEGF mRNA and protein induction by estradiol (E2) in human endometrial fibroblasts. We report here E2 induction of VEGF expression in human venous muscle cells [smooth muscle cells (SMC) from human saphenous veins; HSVSMC] expressing both ER-alpha and ER-beta estrogen receptors. E2 at 10(-9) to 10(-8) M increases VEGF mRNA in HSVSMC in a time-dependent manner (3-fold at 24 h), as analyzed by semiquantitative RT-PCR. This level of induction is comparable with E2 endometrial induction of VEGF mRNA. Tamoxifen and hypoxia also increase HSVSMC VEGF mRNA expression over control values. Immunocytochemistry of saphenous veins and isolated SMC confirms translation of VEGF mRNA into protein. Immunoblot analysis of HSVSMC-conditioned medium detects three bands of 18, 23, and 28 kDa, corresponding to VEGF isoforms of 121, 165, and 189 amino acids. Radioreceptor assay of the conditioned medium produced by E2-stimulated HSVSMC reveals an increased VEGF secretion. Our data indicate that VEGF is E2, tamoxifen, and hypoxia inducible in cultured HSVSMC and E2 inducible in aortic SMC, suggesting E2 modulation of VEGF effects in angiogenesis, vascular permeability, and integrity.     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

The effects of A-ring and D-ring metabolites of estradiol on the proliferation of vascular endothelial cells

The effects of 14 estradiol metabolites on the proliferation of cultured endothelial cells of human umbilical cord veins were examined and compared with that of their parent substance estradiol. The relationship between dosage and effect was tested over the pharmacological concentration range of 10(-8) to 10(-5) M. Estradiol showed a biphasic behaviour, in the form of stimulation at low concentrations and inhibition at the highest concentration. All 10 A-ring metabolites tested stimulated the growth of the endothelial cells at the lower concentrations. At the highest concentration, the 5 A-ring metabolites: 2-hydroxyestrone, 2-hydroxyestradiol, 2-hydroxyestriol, 4-hydroxyestrone and 4-hydroxyestradiol caused significant inhibitions. Except for the 2-hydroxyestradiol, methylation of these metabolites resulted in the loss of the proliferation inhibiting effect. The D-ring metabolites showed no marked effects compared to the A-ring metabolites except for 16alpha-hydroxyestrone which had an inhibiting effect from 10(-7) to 10(-5) M. Our results show that estradiol metabolites can influence the growth of vascular endothelial cells in the concentration range tested. While the antiproliferative action of 2-methoxyestradiol has been known for some time this study is the first to show the potential capacity of non-methylated metabolites of the A-ring metabolism in inhibiting endothelial proliferation. This may open up new clinical pharmacological aspects in the anti-angiogenetic treatment of tumors.     

Diverse effects of vascular endothelial growth factor on human pulmonary endothelial barrier and migration

Increased endothelial permeability is involved in the pathogenesis of many cardiovascular and pulmonary diseases. Vascular endothelial growth factor (VEGF) is a permeability-increasing cytokine. At the same time, VEGF is known to have a beneficial effect on endothelial cells (EC), increasing their survival. Pulmonary endothelium, particularly, may be exposed to higher VEGF concentrations, since the VEGF level is the higher in the lungs than in any other organ. The purpose of this work was to evaluate the effects of VEGF on barrier function and motility of cultured human pulmonary EC. Using transendothelial resistance measurements as an indicator of permeability, we found that 10 ng/ml VEGF significantly improved barrier properties of cultured human pulmonary artery EC (118.6+/-0.6% compared with 100% control, P<0.001). In contrast, challenge with 100 ng/ml VEGF decreased endothelial barrier (71.6+/-1.0% compared with 100% control, P<0.001) and caused disruption of adherens junctions. VEGF at both concentrations increased cellular migration; however, 10 ng/ml VEGF had a significantly stronger effect. VEGF caused a dose-dependent increase in intracellular Ca2+ concentration; however, phosphorylation of myosin light chain was detectably elevated only after treatment with 100 ng/ml. In contrast, 10 ng/ml but not 100 ng/ml VEGF caused a significant increase in intracellular cAMP (known barrier-protective stimulus) compared with nonstimulated cells (1,096+/-157 and 610+/-86 fmol/mg, respectively; P<0.024). Y576-specific phosphorylation of focal adhesion kinase was also stimulated by 10 ng/ml VEGF. Our data suggest that, depending on its concentration, VEGF may cause diverse effects on pulmonary endothelial permeability via different signaling pathways.     

Rat growth hormone-releasing factor stimulates cyclic GMP formation and phosphatidylinositol metabolism in the median eminence.

The effects of rat growth hormone releasing factor (rGRF) on somatostatin (SRIF) secretion, cyclic nucleotide production and phosphatidylinositol metabolism were investigated in the median eminence (ME), using an in vitro system. Medium was discarded and replaced by medium containing various concentrations of rGRF or rGRF plus epinephrine (E, 6 x 10(-7) M). rGRF had no effect on basal or E-stimulated release of cAMP. In the same experiments rGRF markedly stimulated SRIF release. These results suggested that cAMP is not involved in the stimulatory effect of GRF on SRIF release. However, GRF significantly stimulated release of both SRIF and cGMP in a dose-related manner. Maximal stimulation was observed at 10(-10) M GRF (p less than 0.005) which also produces maximal SRIF release. 2'0-monobutyrylguanosine 3'5' cyclic phosphate (mbcGMP, 10(-11) to 10(-10) M) stimulated SRIF release from ME fragments (p less than 0.001 at 10(-10) M) whereas the control, sodium butyrate (10(-6) M), had no effect. GRF caused significant elevation of 30.6% in the concentration of labelled inositol phosphates [( 3H]-IPs) in the ME. These data indicate that GRF stimulation of SRIF release is accompanied by increased cGMP production and phosphatidyl-inositol (PI) metabolism but does not alter cAMP production. Because mbcGMP can directly stimulate SRIF release, we suggest that GRF causes a receptor-mediated increase in the metabolism of phosphatidylinositol and cGMP formation. These actions therefore may be among the early metabolic events in the mechanism of GRF-stimulated SRIF release from the ME.     

Vascular endothelial growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent.

Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell (EC) proliferation and migration in vitro as well as inflammation in vivo. We showed recently that VEGF effect on vascular permeability was dependent on the synthesis of platelet-activating factor (PAF) by EC. Consequently, we sought to evaluate by antisense knockdown of gene expression the contribution of VEGF receptors (Flt-1 and Flk-1) on these events. VEGF (10(-11) to 10(-8) M) elicited a dose-dependent increase of bovine aortic EC proliferation, migration, and PAF synthesis by up to 2.05-, 1.31- and 35.9-fold above basal levels, respectively. A treatment with two modified antisense oligomers (1-5 x 10(-7) M) directed against Flk-1 mRNA blocked by 100, 91, and 85% the proliferation, migration, and PAF synthesis mediated by VEGF, respectively. A treatment with two antisense oligomers directed against Flt-1 mRNA failed to modulate these activities. The use of placenta growth factor (up to 10(-8) M), an Flt-1-specific agonist, induced only a slight increase (0.6-fold) of PAF synthesis. These data illustrate the crucial role of Flk-1 in EC stimulation by VEGF. The capacity to inhibit the protein synthesis of Flt-1 and Flk-1 by antisense oligonucleotides provides a new approach to block VEGF pathological effects in vivo.     

Effect of hypoxia on the release of vascular endothelial growth factor and testosterone in mouse TM3 Leydig cells

Hypoxia has been shown to stimulate the expression of vascular endothelial growth factor (VEGF), which is a major mediator for angiogenesis and vasculogenesis. During hypoxia, VEGF promotes angiogenesis in the testis. However, the effect of VEGF on the steroidogenesis of testosterone and the cell proliferation in Leydig cells is unclear. To assess the effects and the action mechanisms of hypoxia, a mouse TM3 Leydig cell line was employed in the present study. The Leydig cells were incubated in an incubator chamber (95% N2-5% CO2) for 1-24 h. The cultured media were collected and assayed by testosterone RIA and VEGF enzyme immunoassay. 3-(4,50-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide assay was used to detect the proliferation of Leydig cells. The present results showed that the proliferation of Leydig cells was enhanced significantly by hypoxia. The basal VEGF release was increased, and the response of VEGF production to human chorionic gonadotropin (hCG) was also enhanced in hypoxic condition. During hypoxia, administration of hCG or VEGF stimulated proliferation of Leydig cells, but the stimulatory effect was abolished by the administration of anti-VEGF antibody. Higher doses of VEGF stimulated testosterone release in a dose-dependent manner. Administration of anti-VEGF antibody abolished the stimulatory effect of VEGF on testosterone release. These data suggest that hypoxia stimulates cell proliferation and testosterone release in Leydig cells via an increase of VEGF production.     

Endothelin-induced prostacyclin production in rat aortic endothelial cells: role of calcium.

Endothelin (ET) is a potent vasoconstrictor peptide, released from endothelial cells, which is associated with prostaglandin (PG) release. The mechanism by which ET causes the release of PG is not clearly understood. We used rat aortic endothelial cells to investigate the role of calcium (Ca2+) in ET-1-induced prostacyclin (PGI2) release. ET-1 (10(-9) M) produced a significant increase in PGI2 release. Pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of diltiazem (voltage-sensitive L-type calcium channel blocker) produced significant inhibition of ET-1- and PDBu-induced PGI2 release. Inhibition was first noted at 10(-9) M and was complete at 10(-6) M. Conversely, pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of calcium channel blockers (thapsigargin, an intracellular calcium channel blocker or conotoxin, a voltage-sensitive N-type calcium channel blocker) produced no changes on ET-1- or PDBu-induced PGI2 release. These results provide further support for the concept that PKC mediates ET-induced PGI2 release in rat aortic endothelial cells via an increase in intracellular calcium and this increase is due to the influx of extracellular calcium and not to the release of calcium from the sarcoplasmic reticulum.     

Mechanism of action of growth-hormone-releasing hormone in stimulating insulin secretion in vitro from isolated rat islets and dispersed islet cells

Human growth-hormone-releasing hormone [(1-44)NH2] (hGHRH) was a potent stimulus for insulin release from rat islets of Langerhans in vitro; the optimum concentration used was 10(-11) M. The dose response curves for hGHRH effects on insulin secretion were notably different in intact islets of Langerhans compared to cultured dispersed islet cells. Pancreatic islets responded to a very low hGHRH concentration (10(-12) M), but at a higher hGHRH concentration (10(-9) M) no stimulation of insulin release was observed. When somatostatin antiserum was included in the incubation medium, hGHRH (10(-9) M) stimulated insulin release from intact islets. In cultured dispersed islet cells, which are principally insulin-secreting B cells, hGHRH directly and potently stimulated insulin release even at a concentration of 10(-9) M. Addition of somatostatin (10(-7), 10(-8) M) significantly reduced the hGHRH-induced insulin-secretory responses of dispersed islet cells. hGHRH (10(-11)-10(-9) M) raised islet cAMP levels; individually, hGHRH and theophylline exerted positive effects on insulin release, their combined effect was greater than that caused by either one. We conclude that hGHRH directly affects insulin secretion in vitro by a cAMP-dependent mechanism, and that the difference in responses of intact islets versus islet cells to increasing concentrations of hGHRH may be related to hGHRH-induced release of somatostatin in intact rat islets.     

Effects of phorbol ester on GH, TSH and PRL release by superfused rat adenohypophysis

The present study was undertaken to examine the effects of 12-0-tetradecanoyl-phorbol-13-acetate (TPA), one of the potent tumor promoting agents, on GH, TSH and PRL release by rat adenohypophyseal dispersed cells and fragments, using a superfusion technique. TPA (10(-6) to 10(-5) M) stimulated GH release from acutely dispersed rat adenohypophyseal cells. Neither TSH nor PRL was affected, but both were increased by TRH in a dose-dependent fashion (10(-9) to 10(-7) M). In fragments, TPA (10(-8) to 10(-6) M) elicited a dose-related release of GH. Exposure of the fragments to 10(-6) M TPA for 5 min promptly caused a 5-fold increase in GH release which continued for at least 40 min after stopping the stimulation. The addition of somatostatin (SRIF) (10(-7) M) decreased basal GH release and abolished GH release induced by 10(-6) M TPA. In contrast to GH, neither TSH nor PRL release was affected by TPA, but both were stimulated by TRH. These results indicate 1) that GH release is more sensitive to stimulation with TPA in normal rat anterior pituitaries in vitro than the release of TSH and PRL, and 2) that SRIF abolishes TPA-induced GH release.     

4-Hydroxynonenal reduces junctional communication between endothelial cells in culture

The effect of 4-hydroxynonenal (HNE), a lipid peroxidation product, on junctional communication (JC) among cultured vascular endothelial cells was assessed by both study of the transfer of microinjected 6-carboxyfluorescein between neighboring cells and measurement by a "cut-loading and dye transfer" technique. Both methods indicated that at concentrations higher than 10(-9) M and testing times between 6 and 8 h HNE reduces endothelial cell junctional communication. At 10(-8) M, a gradual development of HNE effect appears during 6-8 h of exposure but is followed by a slow recovery completed at 20 h. The reduction in junctional communication is not produced by the inhibition of protein synthesis, as tested by radiolabeled leucine incorporation. The HNE effect might be relevant to pathological processes in which lipid peroxidation is associated with uncontrolled cell proliferation, as in atherogenesis and promotion of carcinogenesis by chronic inflammation.     

Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Deltap85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.     

Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells

Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [³H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two-to threefold) and secretion (1.8-to 2.8-fold) of VEGF reaching maximal levels between 4–8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [³H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma.     

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.     

Production of 5-hydroxyindoleacetic acid from serotonin by cultured endothelial cells

Endothelial cells cells from bovine aorta and human umbilical vein and fibroblasts from human foreskin were cultured and subsequently evaluated for ability to metabolize serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA). Cells were incubated for three hours with 4 X 10(-6) M [14C] 5-HT creatinine sulfate. [14C] 5-HIAA was separated from labeled 5-HT by column chromatography and measured for scintillation counting. Production of 5-HIAA by bovine aorta cells was 39.0+/-7.5 (S.E.M., n=6) nmoles per 10(9) cells per hour. Production of 5-HIAA was markedly inhibited by the presence of 10(-4) M iproniazid (an inhibitor of monoamine oxidase) or 10(-4) M imipramine (an inhibitor of amine transport). 5-HIAA was the only product of 5-HT metabolism detected by thin layer chromatography. Production of 5-HIAA by human umbilical vein endothelial cells was 5.4+/-2.0 nmoles per 10(9) cells per hour (n=5) and by human foreskin fibroblasts was 3.9+/-1.4 nmoles per 10(9) cells per hour (n=5). The results obtained during incubation in the presence and absence of inhibitors indicate that bovine aorta endothelial cells maintained in tissue culture are able to transport serotonin with subsequent production of 5-HIAA. By contrast, human umbilical vein endothelial cells and fibroblasts exhibited relatively low rates of 5-HT uptake and metabolism.