Efflux of taurocholic acid across the blood-brain barrier: interaction with cyclic peptides

The mechanism for the efflux of taurocholic acid (TC) across the blood-brain barrier (BBB) was studied by examining the elimination of [3H]TC after microinjection into the cerebral cortex. The efflux of [3H]TC from the brain was saturable with a Vmax of 15.0 pmol/min/g brain and a Km value of 0.396 nmol/0.2 microl injectate. Efflux was inhibited by cholic acid (CA), a cationic cyclic octapeptide (octreotide; a somatostatin analogue) and an anionic cyclic pentapeptide (BQ-123; an endothelin receptor antagonist), with an IC50 value of 1.09 nmol/0.2 microl injectate, 1.12 nmol/0.2 microl injectate and 0.12 nmol/0.2 microl injectate, respectively. Probenecid (20 nmol/0.2 microl injectate), but not p-aminohippuric acid (10 nmol/0.2 microl injectate), inhibited the brain efflux of [3H]TC. In addition, elimination of [3H]BQ-123 after microinjection was saturable with a Vmax of 20.8 pmol/min/g brain and a Km of 2.92 nmol/0.2 microl injectate; it was also inhibited by TC with an IC50 value of 0.074 nmol/0.2 microl injectate. In contrast, no significant efflux of [14C]octreotide from the brain was observed until 60 min after microinjection. These results suggest that both TC and BQ-123 are transported from the brain to the circulating blood across the blood-brain barrier via specific mechanisms. Although mutual inhibition was observed between TC and BQ-123, kinetic analysis suggested that the two transport systems differ.     

Cold-injury of the cerebral cortex: immunolocalization of cellular proteins and blood-brain barrier permeability studies

The occurrence of blood-brain barrier (BBB) permeability alterations and neovascularization are well documented in the cerebral cortical cold-injury model. This model was used to determine whether the glucose transporter (glutI) protein was present in endothelium of cerebral vessels with breakdown of BBB to protein and when regenerating endothelial cells become immunoreactive for glutI protein. Secondly, the protein products of c-fos and c-jun were localized to determine whether these early immediate genes are activated in this model. Observations were made over a period of 12 hours to 14 days after the cold-injury. Blood-brain barrier permeability was assessed using horseradish peroxidase (HRP) as a tracer. Since HRP may not be able to enter thrombosed vessels within the cold lesion, immunohistochemistry was used to detect extravasation of endogenous serum proteins using antisera to rat serum proteins. The proteins-glut1, GFAP, c-fos and c-jun-were localized by immunohistochemistry. Endothelium of vessels which were permeable to protein, whether in the cold-injury site or in the perilesional area, all contained glut1 protein; hence, the presence of glut1 did not appear to correlate with an intact BBB to protein. An interesting point is that in the process of neovascularization, regenerating endothelial cells become immunoreactive for glut1 at 5 days and this coincides with the presence of tight junctions in these cells. Immunoreactivity for c-fos was observed in regenerating endothelium within the lesion site, in astrocytes, and to a lesser extent in endothelial cells and neurons in the perilesional area. Few astrocytes showed immunoreactivity for c-jun at 4 and 5 days. Possibly, the growth factors generated to promote angiogenesis and repair led to activation of the c-fos gene with deposition of c-fos protein. The results suggest that during nervous system development or endothelial regeneration, the presence of glut1 in cerebral endothelium coincides with the presence of an intact BBB to protein and protein tracers. However, in pathological states presence of glut1 in cerebral endothelium does not appear to correlate with an intact BBB to protein. This model lends itself to the study of angiogenesis and repair processes in the cerebral cortex in an environment unaffected by ischemia and thus the findings may be relevant to traumatic injuries of the human cerebral cortex.     

The effect of photon irradiation on blood-brain barrier permeability to methotrexate in mice

Methotrexate was administered by intraperitoneal injection (100 mg/kg) to unirradiated mice, and to mice receiving varying doses of cranial irradiation. The animals were sacrificed 24 hours after injection, and methotrexate assays were performed on brain tissue. No methotrexate was detected in the brains of the unirradiated animals. Detectable levels of methotrexate were present after 2000 rad cranial irradiation, but not after 500 rad, 1000 rad, or 1500 rad. The implications of these findings are discussed.     

Glycodermorphins: opioid peptides with potent and prolonged analgesic activity and enhanced blood-brain barrier penetration

1. In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O-betaglucosylated analogs ([Ser7-O-betaGlc]dermorphin and [Ser7-O-betaGlc(Ac)4]-dermorphin) and C-alphagalactosylated analogs ([Ala7-C-alphaGal]dermorphin and [Ala7-C-alphaGal(Ac)4]-dermorphin). 2. O- and C-glycosylation of dermorphin halved the peptide affinity for brain mu-opioid receptors and the biological potency in guinea-pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8-40 pmol) and subcutaneously (s.c., 0.5-3 micromol kg(-1)) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5-10 times both biological activity on GPI and mu-receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two-three times lower (s.c.) than dermorphin potency. 3. Blood-Brain Barrier Permeability Index (BBB-PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O-beta-linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT-1 of the endothelial barrier. However the calculated BBB-PI for the C-alphagalactoside was about two times higher than that of the O-betaglucoside, excluding the implication of GLUT-1 that is known to be selective for O-beta-links and preferring for the exose glucose. 4. The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.     

Insight into the regulation by second messenger molecules of the permeability of the blood-brain barrier

Recent advances in our knowledge of the blood-brain barrier have in part been made by studying the properties and function of cerebral endothelial cells in vitro. After an era of working with a fraction, enriched in cerebral microvessels by centrifugation, the next generation of in vitro blood-brain barrier model systems was introduced, when the conditions for routinely culturing the endothelial cells were established. This review summarizes the results obtained mainly from this in vitro approach. Different elements of the intracellular signaling messenger systems have been detected in the course of our studies in the cerebral endothelial cells. It has been shown that the synthesizing enzymes of and substrate proteins for the second messenger molecules are present in the cerebral endothelial cells, and their activity and/or amount can change in pathological circumstances, i.e., during the formation of brain oedema. Pharmacological treatments interfering with the second messenger systems proved to be effective in the prevention of brain oedema formation.     

Cerebral circulation and norepinephrine: relevance of the blood-brain barrier

The systemic administration of norepinephrine has minimal effects on the cerebral circulation, perhaps due to blood-brain barrier mechanisms. To test hypothesis, the cerebrovascular effects of norepinephrine beyond the blood-brain barrier were studied in anesthetized baboons, Intraventricular norepinephrine (40 mug/kg) resulted in significant increases in cerebral blood flow (40%), cerebral oxygen consumption (21%), and cerebral glucose uptake (153%). Intracarotid hypertonic urea opens the blood-brain barrier by osmotic disruption; Consequent to hypertonic urea, the intracarotid infusion of norepinephrine, 50 ng/kg-min, significantly increase cerebral blood flow (49%), cerebral oxygen consumption (21%), and cerebral glucose uptake (76%), It appears probable that the cerebrovascular responses to norepinephrine are dependent on the integrity of the blood-brain barrier; It is likely that the increase in cerebral blood flow, associated with norepinephrine when it bypasses the barrier, is secondary to an increase in cerebral metabolism.     

Passage of cytokines across the blood-brain barrier

One mechanism by which blood-borne cytokines might affect the function of the central nervous system (CNS) is by crossing the blood-brain barrier (BBB) for direct interaction with CNS tissue. Saturable transport systems from blood to the CNS have been described for interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1ra), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Blood-borne cytokines have been shown to cross the BBB to enter cerebrospinal fluid and interstitial fluid spaces of the brain and spinal cord. IL-2 does not cross the BBB by a saturable transport system. The blood-to-brain uptakes of IL-1 alpha, IL-beta, and IL-1ra are interrelated for most brain sites, but the posterior division of the septum shows selective uptake of blood-borne IL-1 alpha. The saturable transport systems for IL-6 and TNF-alpha are distinguishable from each other and from the IL-1 systems. The amount of blood-borne cytokines entering the brain is modest but comparable to that of other water-soluble compounds, such as morphine, known to cross the BBB in sufficient amounts to affect brain function. CNS to blood efflux of cytokines has also been shown to occur, but the mechanism of passage is unclear. Taken together, the evidence shows that passage of cytokines across the BBB occurs, providing a route by which blood-borne cytokines could potentially affect brain function.     

Transport of drugs across the blood-brain barrier

The blood-brain barrier prevents an indifferent medicine existing in the blood to enter also in the brain. This barrier has got an anatomical base: it is first consisting in a cerebrovascular layer of endothelial capillary vessels of the peripheral tissue. It is moreover covered by outgrowths of the flial cells, which are called astrocytes. There are, for that reason, important limits to a size of molecules which can reach the cerebral tissue through a paracellular way (through what is called in English "tight-junctions"). Most medicines must use the transcellular way. Lipophily is necessary to follow that way. Year after year, it appeared, thanks to a comparative study of the substances, that there exists--grosso modo--a positive correlation between the lipophilic level and the permeation-level of a substance in the cerebral tissue. There are, however, several exceptions: it is so that hydrophilic substances, possessing an important nourishing function (such as glucosis, amino-acids) seem to penetrate much more easily than we could expect when we consider their physicochemical characteristics. This is the result of the fact that there exist specifical transport-mechanisms for these substances at the level of the endothelial cell-membranes, allowing the penetration of such substances. There exist, on the contrary, lipophilic components that penetrate the cerebral tissue much less strongly than we should expect. This happens because there also exist pumping-mechanisms at the level of the hemato-encephalic barrier. The concerning substance, which was recently discovered is the "glycoprotein P", which is also responsible for the "multi-drug-resistance" and for the resistance of tumors to cytostatics. This phenomenon relies on a very efficient pumping of substances which have penetrated cells in which this protein expressed itself in the membranous structure. In order to obtain a better understanding of the function of the hemato-encephalic barrier, comprising the transport of medicines, it is most important to have reliable experimental models. It is to that aim that, during former years, the technique of cultivating endothelial cerebrovascular cells was developed. These cells are isolated from brains of calves or rats and, subsequently, cultivated on a laboratory medium; about a week later, they have grown a single and confluent layer. This layer represents a kint of "hemato-encephalic barrier" in vitro, which allows us to study the transfer of substances through the layer and thus also the details concerning the transport mechanisms, as well as the factors influencing the permeability of the cells-layer (for instance the inflammatory stimuli). Concerning the "in vivo" research, the technique of intracerebral microdialysis in lab-animals proved to be very promising. In order to effect this microdialysis, a semipermeable microcannula is introduced in the brain tissue, across which an iso-osmotic liquid is being injected continuously. The substances staying in the interstitial liquid of the cerebral tissue will diffuse under the influence of a concentration gradient, into the dialysing liquid and they will also be ready to be analysed. Thanks to this technique, it is possible to follow, in the same animal, the evolution of the concentration in the brain of a substance which has, for instance been injected in a peripheral region. In this way, we obtain, indirectly and in vivo, informations about the functioning-process of the "hemato-encephalic barrier". We can, moreover, effect measures on a specific spot, for instance in tumoral brain tissue: this allows us to study the influence of specific transport-mechanisms. These rather recent techniques, as well in vitro as in vivo, will allow us, in consequence, to increase, during the next years, our understanding of the way the hemato-encephalic barrier functions as to the transfer of medicines towards the central nervous system. This understanding may lead us to new strategies allowing     

Differential blood-brain barrier breakdown and leucocyte recruitment following excitotoxic lesions in juvenile and adult rats

Acute neuronal degeneration can be induced by intracerebral injections of the glutamate receptor agonists kainic acid (KA) and NMDA (N-methyl-D-aspartate). It is accompanied by an inflammatory response that has not yet been fully investigated. We have previously demonstrated that the juvenile rat brain is more susceptible to an inflammatory challenge when compared to adult rat brain. This study set out to investigate whether this also applied to the inflammatory response associated with acute neuronal degeneration. NMDA and kainic acid were injected into the rat striatum and lesion size, leucocyte recruitment, and blood-brain barrier (BBB) breakdown were assessed after 4, 8, 12, 24, 72, and 168 h. Both NMDA and KA induced lesions of similar volume at either age and apoptotic and necrotic nuclei could be detected. NMDA induced cellular loss by 4 h, whereas KA-injected rats did not show signs of neuronal loss until 8-12 h. The inflammatory response was characterized by an infiltration of neutrophils followed by macrophages. Juvenile rats showed a greater susceptibility to leucocyte recruitment compared to adult rats. BBB breakdown in response to NMDA injection occurred in the absence of cellular recruitment at 4 h in juveniles and was significantly greater in juvenile compared to adult rats at 8 h. BBB breakdown was minimal in KA-injected animals while at 7 days an influx of serum IgG coincided with a loss of astrocytic GFAP staining within the lesion.     

Macromolecular permeability across the blood-nerve and blood-brain barriers

The permeability of insulin (Ins), nerve growth factor (NGF), albumin (Alb), transferrin (Trf), and IgG across the blood-nerve barrier (BNB) and blood-brain barrier (BBB) in normal adult rats was quantified by measuring the (permeability coefficient x surface area) product (PS) with the i.v. bolus-injection technique in the cannulated brachial vein and artery using radioiodinated proteins. The PS values of the BNB for IgG and Alb were low: 0.079 +/- 0.029 x 10(-6) and 0.101 +/- 0.088 x 10(-6) ml.g-1.s-1, (mean +/- SD, respectively). The PS values for NGF and Trf were 16.1-fold and 25.5-fold higher than for Alb. The PS for Ins across the BNB was 33.190 +/- 2.053 x 10(-6) ml.g-1.s-1--a remarkable 329-fold increase compared with Alb. The PS values of the BBB for IgG and Alb in different brain regions were all low, from 0.028 +/- 0.017 to 0.151 +/- 0.035 x 10(-6) ml.g-1.s-1 (mean +/- SD). NGF and Trf had comparable PS values from 13- to 32-fold higher than for Alb, except for the brain stem, where the PS for Trf was 66-fold higher than for Alb. The mean PS for Ins across the BBB ranged from 15.78 +/- 5.45 x 10(-6) ml.g-1.s-1 for the cortex to 22.62 +/- 7.50 x 10(-6) ml.g-1.s-1 for the brain stem--again a remarkable 105- to 390-fold increase relative to Alb. Because reliable PS measurements were obtained for all proteins tested, the BBB and BNB cannot be considered impermeable to proteins--a concept that has plagued brain- and nerve-barrier research. The low PS values for IgG and Alb indicate low rates of transfer; however, Alb, in particular, is the major protein of endoneurial and ventricular fluid, which suggests that these PS values may be significant. Ins had the highest PS values, which likely reflect the mechanism of transport across the barriers--that is, receptor-mediated transport. Because NGF and Trf had PS values 13- to 66-fold higher than for Alb, whether this reflects receptor-mediated uptake, adsorptive-mediated transcytosis, or some other mechanism is unclear. That the PS values for NGF and Trf differ from Alb and IgG clearly suggests, however, a different uptake mechanism. Finally, the remarkably high PS values for Ins across the BBB and BNB identify this protein and its putative receptor on capillary endothelial cells as a potential target for drug delivery into the central and peripheral nervous systems.     

Cysteine and cystine transport at the blood-brain barrier

The nature of cysteine and cystine uptake from the cerebral capillary lumen was studied in the rat using the carotid injection technique, [35S]-Cysteine uptake was readily inhibited by the synthetic amino acid 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), the defining substrate for the leucine-preferring (L) system in the Ehrlich ascites cell. The addition of nonradioactive alanine or serine, representatives of the alanine, serine, and cysteine-preferring (ASC) system, produced no significant decrease in the uptake of cysteine after cysteine transport by the L system was blocked with BCH. This indicated that the major component of cysteine's transport from the brain capillary lumen was by the L system with no detectable uptake of cysteine by the ASC system. No carrier-mediated transport of cystine, the disulfide form of the amino acid, was detected, nor was there any inhibition by cystine of the transport of the neutral amino acid methionine or the basic amino acid arginine. These results suggest that the ASC system, if present, is not quantitatively important for the transport of neutral amino acids from the brain capillary lumen.     

Putrescine-modified nerve growth factor: bioactivity, plasma pharmacokinetics, blood-brain/nerve barrier permeability, and nervous system biodistribution

Previous investigations from our laboratory have demonstrated that the covalent modification of a variety of proteins, including antioxidant enzymes, with the naturally occurring polyamines--putrescine (PUT), spermidine, and spermine--dramatically increases their permeability coefficient-surface area product (PS) at the blood-brain and blood-nerve barriers after parenteral administration. In the present study, we have covalently modified nerve growth factor (NGF) with PUT by targeting carboxylic groups for their graded modification by controlling the ionization of these groups with pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western, and isoelectric focusing analyses demonstrated conversion of NGF to its polyamine-modified derivatives at different pH values. Although the immunoreactivity of PUT-NGF determined by ELISA and western analysis decreased with decreasing pH, the biological activity of PUT-NGF was not affected at any pH as determined by survival and neurite extension of dorsal root ganglia and PC12 cultures. Plasma pharmacokinetics after a single intravenous bolus administration revealed intact PUT-NGF through 10 min and 73-82% intact protein at 15 min. The PS value for PUT-NGF was maximized and the residual plasma volume (Vp) of the protein in the blood vessels minimized when the pH of the modification reaction was >6.4. The biodistribution of PUT-NGF at 15 min showed 22-33% intact protein in different brain regions, which represented 0.4-5.9 ng of PUT-NGF in different brain regions, a physiological dose that is capable of eliciting a bioresponse. The design of this polyamine-modified NGF derivative that has enhanced permeability at the blood-brain and blood-nerve barriers with retained bioactivity may obviate the necessity to create small-molecule mimics of NGF and may be applicable to neurotrophins, engineered multifunctional chimeric neurotrophins, antioxidant enzymes, and other therapeutic proteins with specific clinical application to neurological diseases.     

Convulsions due to increased permeability of the blood-brain barrier in experimental cerebral malaria can be prevented by splenectomy or anti-T cell treatment

Experimental cerebral malaria (ECM) can be induced in C57B1 mice by infection with Plasmodium berghei K173 parasites. Behavioral changes shortly before they die of ECM may reflect disturbance of the integrity of the blood-brain barrier (BBB). Folic acid elicits strong convulsive activity if the permeability of the BBB is increased. Administration of folic acid to mice during development of ECM induced convulsions. Interventions known to prevent fatal outcome from ECM, such as splenectomy or treatment with anti-CD4 or anti-CD8 monoclonal antibodies, also prevented sensitivity to folic acid-induced convulsions. In addition, infected mice with ECM and sensitive to folic acid-induced convulsions, recovered from this sensitivity after treatment with anti-T cell antibodies within 4 h. These data suggest that disturbance of the permeability of the BBB can be reversed and depends on the involvement of T cells.     

Matrix metalloproteinases contribute to the blood-brain barrier disruption during bacterial meningitis

In this study, we investigated the involvement of matrix metalloproteinases (MMPs) in the pathophysiology of bacterial meningitis. By using an enzyme immunoassay, high concentrations of MMP-9 were detected in the cerebrospinal fluid (CSF) of adult patients with bacterial meningitis but not in controls, and in patients with Guillain-Barré syndrome. Moreover, we observed significantly elevated concentrations of the tissue inhibitor of metalloproteinase-1 (TIMP-1) in the CSF of patients with bacterial meningitis, compared with controls. In a rat model of meningococcal meningitis, intracisternal injection of heat-killed meningococci caused a disruption of the blood-brain barrier (BBB), an increase in intracranial pressure, and CSF pleocytosis paralleled by the occurrence of MMP-9 activity in the CSF 6 hours after meningococcal challenge. The MMP inhibitor batimastat (BB-94) significantly reduced the BBB disruption and the increase in intracranial pressure irrespective of the time of batimastat administration (15 minutes before and 3 hours after meningococcal challenge) but failed to significantly reduce CSF white blood cell counts. In conclusion, our results suggest that MMPs are involved in the alterations of BBB permeability during experimental meningococcal meningitis.     

Electron microscopy of the blood-brain barrier in disease

The anatomical site of the blood-brain barrier (BBB) is at the capillary endothelium mainly, with some contribution from astrocytes. Electron microscopic observations of endothelial cells and perivascular astrocytes comprising the BBB in brain edema and other pathological conditions are reviewed in this article. The tight junctions of cerebral endothelial cells open under several conditions such as infusion of hyperosmolar solutions. Pinocytotic vesicles increase under various pathological conditions and fenestrae appear in blood vessels of certain brain tumors and several non-neoplastic lesions. Inflammatory cells penetrate between or through endothelial cells. In long standing lesions, endothelial cells containing various tubular structures such as Weibel-Palade bodies proliferate. Other alterations include surface infoldings of endothelial cells and fluid diffusion through damaged endothelium. Astrocytic alterations include abnormal junctions between astrocytic processes in certain gliomas. In vivo and in vitro studies suggest that astrocytes maintain or develop certain functions of BBB. As the BBB is disrupted, edema fluid infiltrates the brain parenchyma. Because the white matter consists of nerve fibers without demonstrable junctions, it invades between nerve fibers. In the gray matter, expansion of the fluid is limited by complicated anatomical structures. In myelinated nerve fibers, edema fluid accumulates in five separate compartments of extracellular space.     

The blood-brain barrier: morphology, molecules, and neurothelin

The blood-brain barrier (BBB) is a complex structure formed by vascular endothelial cells, which serve to stabilize the homeostasic processes that are essential for neural functioning. The barrier relies on tight junctions between neighboring endothelial cells and a highly restricted passage of blood-borne components through the endothelial lining. Selective transport mechanisms guarantee the essential import and export of metabolites through the BBB into and out of the neural microenvironment. The dual functions of barrier and carrier depend on distinct proteins, some of which have been characterized in detail.     

Anatomy of cerebrospinal fluid system and blood-brain barrier

Baclofen is used for the treatment of post-traumatic spasticity. It carries a risk of overdose as well as of an acute withdrawal syndrome. We report two cases of severe hypertonia and hyperthermia (> 42 degrees C), occurring after accidental discontinuation of intrathecal infusion of baclofen. Both hypertonia and hyperthermia ceased when administration of baclofen was resumed. In parallel, the patients developed transient life-threatening alterations of hepatic (cytolysis), haematologic (coagulopathy) and cardiorespiratory functions for some days. It is concluded that the occurrence of such a withdrawal syndrome should be prevented, especially in patients with chronic intrathecal administration and first symptoms should be recognized without delay. Relationships with other malignant hyperthermias are discussed.     

Effect of global system for mobile communication (GSM) microwave exposure on blood-brain barrier permeability in rat

Reliability of nursing observations often is estimated using Cohen's kappa, a chance-adjusted measure of agreement between observer RNs. However, use of kappa as an omnibus measure sometimes can be misleading. In a study partly designed to describe the frequency and reliability of nursing diagnoses in long-term care facilities, 360 residents each were assessed independently by two registered nurses, and kappa and observed proportion of agreement were calculated as estimates of reliability. For some diagnoses we observed high proportions of agreement, yet paradoxically low kappa values. This article presents an in-depth statistical analysis to resolve this paradox. Results from our analysis also suggest means for planning improvements in the diagnostic performance of participating RNs. Consequently, our approach can be used in similar studies of diagnosis reliability to enhance nursing research, education, and practice.     

Foscarnet penetrates the blood-brain barrier: rationale for therapy of cytomegalovirus encephalitis

Hypertension is a major complication of rHuEPO therapy in hemodialysis (HD) patients. We have previously reported that patients receiving rHuEPO intravenously (i.v.) had higher mean arterial pressure (MAP) and plasma endothelin-1 (ET-1) levels than those in which the hormone was administered subcutaneously (s.c.). To test whether the increased serum ET-1 levels associated with i.v. rHuEPO administration are the result of a direct effect of the hormone on ET-1 release by the endothelial cells (EC), we examined the effects of rHuEPO in vitro. Bovine pulmonary artery endothelial cells (BPAEC) were exposed to doses of rHuEPO of 0.8; 1.6; 3.3 and 6.6 U/ml. A 24 hour-time course showed maximal ET-1 production at 12 hours for all the doses tested. A significant increase in cell proliferation over controls was observed at 24 hours, for all rHuEPO doses, and no correlation was found between ET-1 values and cell proliferation. Inhibition of protein synthesis by cycloheximide (10 micrograms/ml) abolished the stimulation of ET-1 release by rHuEPO. Thrombin (4 U/ml) and angiotensin II (10(-7) M), two potent stimulators of ET-1 release, had additive effects to those of rHuEPO. Specific thrombin and angiotensin II antagonists blocked these additive effects, reducing ET-1 release to the level of rHuEPO stimulation alone. In summary, rHuEPO stimulates vascular EC in culture to increase ET-1 release through an increase in synthesis and in a time dependent fashion. The routes of stimulation seem to differ from other known ET-1 secretogoges. Our data also confirm a significant mitogenic effect of rHuEPO on the endothelial cell.