Oxygen transport and consumption by suspended cells in microgravity: a multiphase analysis

A rotating bioreactor for the cell/tissue culture should be operated to obtain sufficient nutrient transfer and avoid damage to the culture materials. Thus, the objective of the present study is to determine the appropriate suspension conditions for the bead/cell distribution and evaluate oxygen transport in the rotating wall vessel (RWV) bioreactor. A numerical analysis of the RWV bioreactor is conducted by incorporating the Eulerian-Eulerian multiphase and oxygen transport equations. The bead size and rotating speed are the control variables in the calculations. The present results show that the rotating speed for appropriate suspensions needs to be increased as the size of the bead/cell increases: 10 rpm for 200 microm; 12 rpm for 300 microm; 14 rpm for 400 microm; 18 rpm for 600 microm. As the rotating speed and the bead size increase from 10 rpm/200 microm to 18 rpm/600 microm, the mean oxygen concentration in the 80% midzone of the vessel is increased by approximately 85% after 1-h rotation due to the high convective flow for 18 rpm/600 microm case as compared to 10 rpm/200 microm case. The present results may serve as criteria to set the operating parameters for a RWV bioreactor, such as the size of beads and the rotating speed, according to the growth of cell aggregates. In addition, it might provide a design parameter for an advanced suspension bioreactor for 3-D engineered cell and tissue cultures.     

Dehydroepiandrosterone stimulates nitric oxide release in vascular endothelial cells: evidence for a cell surface receptor

Dehydroepiandrosterone (DHEA) improves vascular function, but the mechanism of this effect is unclear. Since nitric oxide (NO) regulates vascular function, we hypothesized that DHEA affects the vasculature by increasing endothelial NO production. Physiological concentrations of DHEA stimulated NO release from intact bovine aortic endothelial cells (BAEC) within 5min. This effect was mediated by activation of endothelial nitric oxide synthase (eNOS) in BAEC and human umbilical vein endothelial cells (HUVEC). Dehydroepiandrosterone increased cyclic GMP (cGMP) levels in BAEC, consistent with its effect on NO production. Albumin-conjugated DHEA also stimulated NO release, suggesting that DHEA stimulates eNOS by a plasma membrane-initiated signal. Tamoxifen blocked estrogen-stimulated NO release from BAEC, but did not inhibit the DHEA effect. Pertussis toxin abolished the acute effect of DHEA on NO release. Dehydroepiandrosterone had no effect on intracellular calcium fluxes. However, inhibition of tyrosine kinases or the mitogen-activated protein (MAP) kinase kinase (MEK) blocked NO release and cGMP production in response to DHEA. These findings demonstrate that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells, by a plasma membrane-initiated mechanism. This action of DHEA is mediated by a steroid-specific, G-protein coupled receptor, which activates eNOS in both bovine and human cells. The release of NO is independent of intracellular calcium mobilization, but depends on tyrosine- and MAP kinases. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for DHEA.     

Production of somatic embryos in a helical ribbon impeller bioreactor

Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11-L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures ( approximately 8-13 SE . mL(-;1)). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate ( approximately 0.05 VVM, k(L) a approximately 6.1 h(-;1)) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO ( approximately 60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (-5-10%) inhibited biomass and SE production. The best production of SE ( approximately 44 SE . mL(-1) or approximately 757 SE . g dw(-1) . d(-1)) was achieved by operating the bioreactor at 60 rpm while controlling DO at approximately 20%by surface oxygenation only (0.05 VVM, k(L) a approximately 1.4 h(-;1)). This production was found to be a biomass production/growth-associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. (c) 1994 John Wiley & Sons, Inc.     

Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase

Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+]i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+]i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.     

Anthocyanin production from Vaccinium pahalae: limitations of the physical microenvironment.

Physical microenvironmental parameters conducive to production of flavonoids in vitro from continuous Vaccinium pahalae suspension cultures were examined first in shake flask culture experiments, and results were used to guide adaptations of a bioreactor production environment. Anthocyanin pigments were primarily concentrated in smaller aggregates up to 519 microm diameter. Agitation at 150 rpm and routine use of a mechanical scraper to periodically retrieve cells thrown out of solution was sufficient to keep productive plant cell aggregates in bioreactor suspension. A set up for enhanced irradiance with mercury lamps providing an average of 240 micromol m(-2) s(-1) PPF at the inner surface of the bioreactor vessel was required to sustain anthocyanin productivity through a 10-day production cycle.     

Production of extracellular exoinulinase from Kluyveromyces marxianus YS-1 using root tubers of Asparagus officinalis

Root tubers of Asparagus officinalis were used as a source of raw inulin for the production of exoinulinase (EC 3.2.1.7) from Kluyveromyces marxianus YS-1. Root extract prepared at 10kg/cm2 pressure for 10min showed maximum inulinase production. Medium components and process parameters were standardized to improve the enzyme production. Inulinase yield of 40.2IU/mL in a medium containing raw inulin (3.5%), beef extract (2%), SDS (0.001%), Mn2+ (2.0mM), Mg2+ (1.5mM), Co2+ (2mM) and pH 6.5 has been obtained under agitation (150rpm) after 60h of incubation at 30 degrees C at shake flask level. After optimization, the enzyme production was 4.8 times more than the basal medium. To test the feasibility of raw inulin from A. officinalis for the production of inulinase, trials were also made in a bioreactor (1.5L). Inulinase activity of 50.2IU/mL was obtained from raw inulin (4.0%) under agitation (200rpm) and aeration (0.75vvm) at 30 degrees C after 60h of fermentation. Inulinase yield in bioreactor was almost six times higher than the basal medium used initially in shake flask.     

胰岛素促进血管内皮细胞产生一氧化氮的实验研究

目的：探讨胰岛素对血管内皮细胞增殖、NO产生和NOS基因表达的影响。方法：培养牛主动脉内皮细胞,测定培养上清液中NO氧化产物NO2^－的水平并应用定量RT－PCR技术检测内皮细胞NOS mRNA的表达水平。结果：①胰岛素对大血管内皮细胞无细胞毒作用,也不影响细胞增殖;②在1－15μg/ml浓度范围内,胰岛素加强内皮细胞释放NO,且呈剂量依赖的方式,NOS特异性抑制剂L－NAME可阻抑之;③胰岛素轻度增加NOS mRNA表达水平,但无统计学意义。结论：胰岛素既不影响大血管内皮细胞增殖,也不影响内皮细胞NOS mRNA表达水平,但以剂量依赖的方式加强内皮细胞产生NO,推测其诱导NO产生的机制可能是通过酶活性的诱导,加速NO的合成。     

Studies of chondrogenesis in rotating systems

A great deal of energy has been exerted over the years researching methods for regenerating and repairing bone and cartilage. Several techniques, especially bone implants and grafts, show great promise for providing a remedy for many skeletal disorders and chondrodystrophies. The bioreactor (rotating-wall vessel, RWV) is a cell culture system that creates a nurturing environment conducive to cell aggregation. Chondrocyte cultures have been studied as implants for repair and replacement of damaged and missing bone and cartilage since 1965 [Chesterman and Smith, J Bone Joint Surg 50B:184–197, 1965]. The ability to use large, tissue-like cartilage aggregates grown in the RWV would be of great clinical significance in treating skeletal disorders. In addition, the RWV may provide a superior method for studying chondrogenesis and chondrogenic mutations. Because the RWV is also reported to simulate many of the conditions of microgravity it is a very useful ground-based tool for studying how cell systems will react to microgravity. © 1993 Wiley-Liss, Inc.     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

Human free apolipoprotein A-I and artificial pre-beta-high-density lipoprotein inhibit eNOS activity and NO release

Little is known about the effects of human free apolipoprotein A-I (Free-Apo A-I) and pre-beta-high density lipoprotein (pre-beta-HDL) on the endothelium function. In this study, we have investigated the effects of Free-Apo A-I and artificial pre-beta-HDL on endothelial NO synthase (eNOS) activity and on NO production by endothelial cells. Free-Apo A-I drastically inhibited NO production in human umbilical cord vein endothelial cells (HUVECs) and eNOS activity in bovine aortic endothelial cells (BAECs). Pre-beta-HDL and serum from human apolipoprotein A-I transgenic rabbits inhibited eNOS activity in BAECs but HDL3 did not. Free-Apo A-I displaced eNOS from BAEC plasma membrane towards intracellular pools without affecting eNOS activity and eNOS mass in BAEC crude homogenates. Free-Apo A-I and HDL3 did not decrease either caveolin bound to BAEC plasma membrane or caveola cholesterol content. As previously described, we showed that HDL3 directly induced endothelium-dependent relaxation of rings from rat aorta. We observed that pre-beta-HDL significantly decreased endothelium-dependent relaxation of rat aortic rings ex vivo.     

Regulation of NO from endothelial cells by the decrease of cellular cAMP under arsenite exposure

In an attempt to delineate the direct effect of arsenite-induced endothelial dysfunction on nitric oxide (NO) production, confluent bovine aortic endothelial cells (BAEC) were incubated with arsenite, and endothelial NO synthase expression and NO production were measured. Exposure of arsenite decreased NO production for up to 24 h. This decrease was accompanied by decreases in cAMP, protein kinase A (PKA) activity, and furthermore, significant reduction of pCREB. In conclusion, this study is the first to demonstrate that exposure of arsenite decreases NO production by a reduction of pCREB and PKA activity that may be mediated by cAMP, leading to endothelial dysfunction.     

Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress.

Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.     

A NMR-compatible and reduced gravity simulation based (NRG) bioreactor for on-line monitoring cell culture metabolism

We developed a NMR-compatible microgravity-based bioreactor (NRG[R]) that offers the advantage of an analytical non-invasive approach associated to the effects of an optimized suspension culture. The simulated microgravity conditions reached in the bioreactor are analogous to those of commercial apparatus like the Rotating Wall Vessel (RWV) system. The faster proliferation of endothelial cells cultured in the NRG bioreactor (doubling time : 28 +/- 1.7 vs. 43 +/- 5.6 h of the control grown in RWV) are attributed to different oxygenation conditions and medium wash out.     

Cross-linking density alters early metabolic activities in chondrocytes encapsulated in poly(ethylene glycol) hydrogels and cultured in the rotating wall vessel

In designing a tissue engineering strategy for cartilage repair, selection of both the bioreactor, and scaffold is important to the development of a mechanically functional tissue. The hydrodynamic environment associated with many bioreactors enhances nutrient transport, but also introduces fluid shear stress, which may influence cellular response. This study examined the combined effects of hydrogel cross-linking and the hydrodynamic environment on early chondrocyte response. Specifically, chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels having two different cross-linked structures, corresponding to a low and high cross-linking density. Both cross-linked gels yielded high water contents (92% and 79%, respectively) and mesh sizes of 150 and 60 A respectively. Cell-laden PEG hydrogels were cultured in rotating wall vessels (RWV) or under static cultures for up to 5 days. Rotating cultures yielded low fluid shear stresses (< or = 0.11 Pa) at the hydrogel periphery indicating a laminar hydrodynamic environment. Chondrocyte response was measured through total DNA content, total nitric oxide (NO) production, and matrix deposition for glycosaminoglycans (GAG). In static cultures, gel cross-linking had no effect on DNA content, NO production, or GAG production; although GAG production increased with culture time for both cross-linked gels. In rotating cultures, DNA content increased, NO production decreased, and overall GAG production decreased when compared to static controls for the low cross-linked gels. For the high cross-linked gels, the hydrodynamic environment had no effect on DNA content, but exhibited similar results to the low cross-linked gel for NO production, and matrix production. Our findings demonstrated that at early culture times, when there is limited matrix production, the hydrodynamic environment dramatically influences cell response in a manner dependent on the gel cross-linking, which may impact long-term tissue development.     

The use of microcarrier beads in the production of endothelium-derived relaxing factor by freshly harvested endothelial cells.

This study is concerned with the use of freshly harvested bovine endothelial cells attached to microcarrier beads in the production of the endothelium-derived relaxing factor (EDRF). The results are compared to production of EDRF by endothelial cells grown in tissue cultures. We found that freshly harvested cells attach themselves to microcarrier beads within minutes. This results in large surface/area volume ratio and permits superfusion of cells suspension on a filter (pore size of 25-30 microns), resulting in cell free filtrate. When superfusing an endothelium-deprived pulmonary artery strip, the effluent causes relaxation; the response depends on the number of superfused endothelial cells. The number of viable freshly harvested cells attached to microcarrier beads in 5 ml Krebs-Henseleit solution is small (30%), as compared to almost 100% for cultured cells. Despite this difference, percent relaxation induced for the same number of viable cells is identical for both groups. Scanning electromicrographs confirm anchorage of endothelial cells to microcarrier beads. While cultured cells cover the entire surface and are individually attached, freshly harvested cells are anchored as cell aggregates leaving some of the surface free. Attachment of freshly harvested endothelial cells to microcarrier beads offers an alternative for the study of the role of endothelial cells in the production of vasoactive substances.     

Chk1 and Hsp90 cooperatively regulate phosphorylation of endothelial nitric oxide synthase at serine 1179

The effects of DNA damage on NO production have not been completely elucidated. Using ultraviolet (UV) irradiation as a DNA-damaging agent, we studied its effect on NO production in bovine aortic endothelial cells (BAEC). UV irradiation acutely increased NO production, the phosphorylation of endothelial NO synthase (eNOS) at serine 1179, and eNOS activity. No alterations in eNOS expression nor phosphorylation at eNOS Thr⁴⁹⁷ or eNOS Ser¹¹⁶ were found. SB218078, a checkpoint kinase 1 (Chk1) inhibitor, inhibited UV-irradiation-stimulated eNOS-Ser¹¹⁷⁹ phosphorylation and NO production. Similarly, ectopic expression of small interference RNA for Chk1 or a dominant-negative Chk1 repressed the UV-irradiation stimulatory effect, whereas wild-type Chk1 increased basal eNOS-Ser¹¹⁷⁹ phosphorylation. Purified Chk1 directly phosphorylated eNOS Ser¹¹⁷⁹ in vitro. Confocal microscopy and coimmunoprecipitation studies revealed a colocalization of eNOS and Chk1. In basal BAEC, heat shock protein 90 (Hsp90) predominantly interacted with Chk1. This interaction, which decreased significantly in response to UV irradiation, was accompanied by increased interaction of Hsp90 with eNOS. The Hsp90 inhibitor geldanamycin attenuated UV-irradiation-stimulated eNOS-Ser¹¹⁷⁹ phosphorylation by dissociating Hsp90 from eNOS. UV irradiation and geldanamycin did not alter the interaction between eNOS and Chk1. Overall, this is the first study demonstrating that Chk1 directly phosphorylates eNOS Ser¹¹⁷⁹ in response to UV irradiation, which is dependent on Hsp90 interaction.     

B56α subunit of protein phosphatase 2A mediates retinoic acid-induced decreases in phosphorylation of endothelial nitric oxide synthase at serine 1179 and nitric oxide production in bovine aortic endothelial cells

We previously showed that all-trans retinoic acid (atRA) decreased nitric oxide (NO) production through Akt-mediated decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS-Ser¹¹⁷⁹) in bovine aortic endothelial cells (BAEC). Since protein phosphatase 2A (PP2A) was also reported to decrease eNOS-Ser¹¹⁷⁹ phosphorylation, we investigated using BAEC whether PP2A mediates atRA-induced eNOS-Ser¹¹⁷⁹ dephosphorylation and subsequent decreased NO production. Treatment with okadaic acid (5 nM), a selective PP2A inhibitor, or ectopic expression of small interference RNA (siRNA) of PP2A catalytic subunit α (PP2A Cα) significantly increased eNOS-Ser¹¹⁷⁹ phosphorylation and NO production. Each treatment also significantly reversed atRA-induced observed effects, suggesting a role for PP2A. We also found that atRA significantly increased cellular PP2A activity. However, Western blot analysis revealed that atRA did not increase the expression of PP2A Cα, although it significantly increased the level of B56α of PP2A regulatory B subunit (PP2A B56α), but not PP2A B55α and PP2A B56δ. Real-time PCR assay confirmed a significant increase in PP2A B56α mRNA expression in atRA-treated cells. Ectopic expression of siRNA of PP2A B56α significantly reversed atRA-induced inhibitory effects on eNOS-Ser¹¹⁷⁹ phosphorylation and NO production, suggesting a role for PP2A B56α. Our study demonstrates for the first time that atRA decreases eNOS-Ser¹¹⁷⁹ phosphorylation and NO release at least in part by increasing PP2A B56α-mediated PP2A activity in BAEC.     

5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures.

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.