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Am J Physiol Heart Circ Physiol 278: H233-H238, 2000;
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Vol. 278, Issue 1, H233-H238, January 2000

Cross talk of shear-induced production of prostacyclin and nitric oxide in endothelial cells

Tomohiro Osanai, Norio Fujita, Naoto Fujiwara, Takao Nakano, Koki Takahashi, Weiping Guan, and Ken Okumura

Second Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that vessel homeostasis is maintained through the cross talk of shear-induced production of prostacyclin and nitric oxide (NO). Confluent human umbilical vein endothelial cells (HUVEC) were exposed to fluid shear stress at 15 dyn/cm2 using a cone-plate device, and the concentrations of 6-keto-PGF1alpha and NO metabolites (nitrate and nitrite) in the medium were measured with radioimmunoassay and the Greiss method, respectively. Compared with static control, shear stress increased cumulative prostacyclin production by twofold after 90 min of exposure. Inhibition of NO synthase enhanced flow-induced prostacyclin production by twofold without affecting the baseline production. Guanylyl cyclase inhibitor enhanced flow-induced prostacyclin production to the same degree. In contrast, a stable agonist of cGMP attenuated the rapid early phase of flow-dependent prostacyclin production. Shear-induced NO metabolite production was unaffected even after indomethacin inhibited prostacyclin production. We conclude that NO shows an inhibitory effect on prostacyclin production under shear stress and that vessel homeostasis may be maintained through an increase in prostacyclin production when NO synthesis is impaired in endothelial cells.

shear stress; guanosine 5'-triphosphate-binding protein; guanosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIAL CELLS in normal blood vessels secrete prostacyclin and nitric oxide (NO), and these compounds synergistically contribute to vessel homeostasis by inhibiting vascular smooth muscle tone (31, 35) and growth (12, 18), platelet aggregation (16), leukocyte adhesion to endothelium (34, 35, 37), and susceptibility to thrombosis (26). The passage of blood in the vessels generates hemodynamic forces and regulates the function of endothelial cells lining the intimal surface of the vasculature (19). Shear stress stimulates the synthesis and secretion of various bioactive molecules such as prostacyclin, NO, tissue plasminogen activator, and platelet-derived growth factor (9, 11, 24), inhibits cell proliferation (1), initiates intracellular signaling (25, 28, 30, 32), and induces elongation of endothelial cells in the direction of the flow through cytoskeletal rearrangement (8, 38). Recent studies on the mechanism for flow-induced enhancement of prostacyclin and NO production showed that it is mediated by the activation of GTP-binding proteins, as demonstrated by their inhibition by guanosine 5'-O-(2-thiodiphosphate), a nonhydrolyzable analog of GDP (3, 20). Flow-induced prostacyclin production is associated with the activation of phospholipase A2, which is mediated by pertussis toxin (PTX)-sensitive GTP-binding proteins Gi-3, and the subsequent release of arachidonic acid from membrane phospholipids (3, 6, 11, 14). In contrast, NO production is mediated by the activation of phospholipase C linked to PTX-insensitive GTP-binding proteins, which rapidly stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (5, 28, 33).

Although blood flow is a critical regulator of endothelial prostacyclin and NO production in the physiological context of the blood vessel, the cross talk of these compounds has not been investigated under shear conditions. In clinical studies, many lines of evidence indicate that NO production is impaired in patients with atherosclerosis, on the basis of the responses of regional blood flow and blood concentration of cGMP to the agents that stimulate or inhibit endogenous NO production (15). In contrast, urinary 2,3-dinor-6-keto-PGF1alpha , a stable metabolite of prostacyclin, is not reduced, even in patients with manifest atherosclerotic diseases (10). The possibility arises that vessel homeostasis may be maintained through the cross talk of production of prostacyclin and NO in vivo. Thus this study was designed to elucidate the cross talk between the flow-induced production of prostacyclin and NO in endothelial cells in vitro. Furthermore, we investigated the intracellular mechanism for this effect.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Cell culture media were purchased from Kurabo. Trypsin (0.05%)-EDTA (0.02%), petri dishes, DMEM, and PBS were from GIBCO (Grand Island, NY). A tritiated 6-keto-PGF1alpha tracer was purchased from New England Nuclear (Boston, MA). 6-Keto-PGF1alpha antibody and standard were provided by Ono Pharmaceutical. The nitrate and nitrite assay kit was purchased from Cayman Chemical (Ann Arbor, MI). Indomethacin, NG-nitro-L-arginine methyl ester (L-NAME), methylene blue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), guanosine 5'-O-3-(thiotriphosphate) (GTPgamma S), and all other reagents were of the finest grade available (Sigma Chemical, St. Louis, MO).

Cell culture. Primary human umbilical vein endothelial cells (HUVEC) were harvested from umbilical cords. For removal of the endothelial cells, the veins were cannulated, rinsed with 100 ml of PBS, filled with 0.03% collagenase in DMEM, and incubated for 30 min at room temperature. After incubation, the enzyme solution was flushed through the cord with 100 ml of PBS and the effluent was collected and centrifuged at 100 g for 10 min. The cell pellet was resuspended in HuMedia supplemented with 2% fetal bovine serum, 10 ng/ml recombinant epidermal growth factor, 1 µg/ml hydrocortisone, 5 ng/ml recombinant fibroblast growth factor, and 10 µg/ml heparin (complete medium) and seeded on 75-cm2 flasks. Media in all cultures were renewed every 2-3 days. The culture supplemented with various chemicals was incubated at 37°C for 30 min before exposure to shear stress.

Flow method. Confluent HUVEC, last fed with the complete growth medium, were exposed to a fluid shear stress with the use of a cone-plate viscometer specifically designed to accept standard tissue culture plates (21). Shear stress magnitude (= µw/alpha ) was computed by using relations previously derived by Sdougos et al. (36), where µ is fluid viscosity, w is rotational velocity, and alpha  is the cone angle. In the steady laminar mode, a cone of 1° angle spinning at 4 and 6.7 revolutions/s was used to achieve an average shear stress magnitude of 15 and 25 dyn/cm2. The cells inside the viscometer were kept at a temperature of 37°C in a humidified atmosphere with 5% CO2. Viability of the cells exposed to shear stress was assessed by the measurement of protein content of the dishes and the staining with trypan blue.

Protocol. HUVEC in 75-cm2 flasks were subcultured with trypsin (0.05%)-EDTA (0.02%) into four 100-mm petri dishes and were maintained in the complete medium. Confluent monolayers in petri dishes were gently washed three times with PBS, and the medium was replaced with 5 ml of serum-free DMEM. One monolayer of the petri dish was kept under a static condition (control), and the others were exposed to shear stress (15 and 25 dyn/cm2) for 90 min at 37°C under 5% CO2. To examine the reproducibility of shear-induced production of prostacyclin and NO, we further subjected the cells already exposed to shear stress at 15 dyn/cm2 to the same shear stress for another 90-min period after a 30-min static interval. The medium (100 µl) was taken from the cultures with and without shear stress conditions for measurements of 6-keto-PGF1alpha and nitrite at 20, 40, 60, and 90 min after initiation of the study. In the other set of experiments, the confluent monolayer was preincubated with 10-4 M L-NAME, 10-5 M indomethacin, 20 µM methylene blue, or 2 mM 8-BrcGMP for 30 min and the medium was replaced with 5 ml of fresh medium. These cells were subjected to shear stress at 15 dyn/cm2 for 90 min, and the medium (100 µl) was taken for measurements of 6-keto-PGF1alpha and nitrite.

Studies on the site(s) of action of cGMP were carried out in a static condition. HUVEC on 24-well plates were preincubated with GTPgamma S, a nonhydrolyzable form of GTP, at 0.2 mM for 4 h, washed three times with PBS, and then incubated with 200 µl of DMEM in the presence or absence of 8-BrcGMP (2 mM). After 90 min, the medium (100 µl) was taken for measurement of 6-keto-PGF1alpha . In another set of experiments, HUVEC on 24-well plates were washed three times with PBS and then incubated in serum-free DMEM supplemented with ionomycin at 1 µg/ml, 8-BrcGMP at 2 mM, or both. The medium was taken 90 min after the onset of incubation for the subsequent measurement of 6-keto-PGF1alpha .

PG measurement. The concentration of 6-keto-PGF1alpha was measured by radioimmunoassay according to the method of Inagawa et al. (17) with the use of a specific antibody to 6-keto-PGF1alpha . The cross-reaction rate of the 6-keto-PGF1alpha antiserum was 9.1% for PGE2, 5.0% for PGF2alpha , and undetectable for thromboxane B2. Cellular protein was determined by the Bradford method. The amount of 6-keto-PGF1alpha secreted from the cells was expressed as picograms per microgram of protein.

Assay for NO metabolite level in conditioned medium. Nitrate and nitrite content in the conditioned medium was measured to determine the enzymatic production of NO from L-arginine by cultured cells (13). After conversion of nitrate to nitrite in the presence of nitrate reductase and cofactor, aliquots of conditioned medium were mixed with an equal volume of Greiss reagent [1:1 (vol/vol) mixture of 1% sulfanilamide and 0.1% naphthylethylenediamine in 2% phosphoric acid]. After color developed, the optical density was determined at 540 nm. The concentration of unknowns was determined by comparison with values from a standard curve obtained with sodium nitrite.

Statistics. Results are presented as means ± SE. Differences in time-dependent prostacyclin production were analyzed by two-way ANOVA. Comparison of prostacyclin production among conditions with GTPgamma S, ionomycin, and 8-BrcGMP was carried out by one-way ANOVA. Differences were considered significant at an error probability of <0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Viability of cells. Protein content was 149.6 ± 16.0 µg/dish under static control conditions and 137.6 ± 15.2 and 142.2 ± 14.2 µg/dish under shear stress conditions at 15 and 25 dyn/cm2, respectively, after a period of 90 min [P = not significant (NS)]. Successive exposure to shear stress at 15 dyn/cm2 also did not affect the protein content. Treatment with indomethacin, L-NAME, methylene blue, or 8-BrcGMP had no influence on the protein content after 90-min exposure to shear stress. Trypan blue-positive cells were undetectable in both static control and shear conditions.

Prostacyclin production. As shown in Fig. 1, 6-keto-PGF1alpha in the conditioned medium accumulated in a time-dependent manner in a static condition and the level at 90 min was 5.7 ± 1.9 pg/µg protein. Sudden onset of laminar shear stress at 15 dyn/cm2 after culture in a static condition caused a rapid and marked increase in prostacyclin production, which was followed by a sustained phase. Shear-induced increase in prostacyclin production was time dependent, and the level after 90 min of exposure was twofold greater than that under a static control condition. Further exposure of the cells for another 90-min period to the same shear at 15 dyn/cm2 after the 30-min static interval caused a similar increase in prostacyclin production (12.1 ± 1.5 pg/µg protein after initial shear and 11.4 ± 3.0 pg/µg protein after 2nd shear, n = 3, P = NS). Prostacyclin production was 11.5 ± 1.3 pg/µg protein under shear at 15 dyn/cm2 and 22.5 ± 2.1 pg/µg protein under shear at 25 dyn/cm2.


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  		Fig. 1.   Effect of NG-nitro-L-arginine methyl ester (L-NAME) on shear (15 dyn/cm2)-induced prostacyclin production in human umbilical vein endothelial cells. Shear (+), cells subjected to shear stress at 15 dyn/cm2 for 90 min at 37°C under 5% CO2; shear (-), cells kept under a static condition for 90 min at 37°C under 5% CO2. Data are means ± SE for 9 experiments in duplicate. * P < 0.05 by 2-way ANOVA.

Inhibition of NO synthase with 10-4 M L-NAME enhanced flow-induced prostacyclin production (P < 0.05 by 2-way ANOVA) twofold without affecting the baseline production, and the levels after 90 min in cultures exposed to shear in the absence (Fig. 1) and presence of L-NAME were 11.2 ± 1.6 and 24.5 ± 5.8 pg/µg protein, respectively. Treatment with methylene blue at 20 µM also enhanced the increase in prostacyclin production twofold, just as seen with L-NAME (Fig. 2). In contrast, 8-BrcGMP at 2 mM attenuated the rapid early phase of flow-dependent prostacyclin production, resulting in slowly progressive production (Fig. 3). However, the levels after 90 min in cultures exposed to shear in the absence and presence of 8-BrcGMP showed no significant difference (10.8 ± 1.5 vs. 9.7 ± 4.0 pg/µg protein, P = NS).


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  		Fig. 2.   Effect of methylene blue (MB) on shear (15 dyn/cm2)-induced prostacyclin production in human umbilical vein endothelial cells. Shear (+), cells subjected to shear stress at 15 dyn/cm2 for 90 min at 37°C under 5% CO2; shear (-), cells kept under a static condition for 90 min at 37°C under 5% CO2. Data are means ± SE for 8 experiments in duplicate. * P < 0.05 by 2-way ANOVA.



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  		Fig. 3.   Effect of 8-bromo-guanosine 3',5'-cyclic monophosphate (8-BrcGMP) on shear (15 dyn/cm2)-induced prostacyclin production in human umbilical vein endothelial cells. Shear (+), cells subjected to shear stress at 15 dyn/cm2 for 90 min at 37°C under 5% CO2; shear (-), cells kept under a static condition for 90 min at 37°C under 5% CO2. Data are means ± SE for 7 experiments in duplicate.

Figure 4 illustrates the effect of 8-BrcGMP on GTPgamma S-induced prostacyclin production in a static condition. GTPgamma S (0.2 mM) used as the activator of GTP-binding proteins enhanced the 90-min production of prostacyclin by twofold (6.4 ± 0.7 vs. 11.3 ± 0.9 pg/µg protein, P < 0.05), whereas cotreatment with 8-BrcGMP (2 mM) abolished the enhanced production of prostacyclin by GTPgamma S. In contrast, as shown in Fig. 5, ionomycin enhanced the prostacyclin production by eightfold, whereas cotreatment with 8-BrcGMP did not affect ionomycin-induced prostacyclin production.


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  		Fig. 4.   Effect of 8-BrcGMP on guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S)-induced prostacyclin production in human umbilical vein endothelial cells. Data are means ± SE for 4 experiments in duplicate. * P < 0.05 by 1-way ANOVA; ns, not significant.



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  		Fig. 5.   Effect of 8-BrcGMP on ionomycin (Iono)-induced prostacyclin production in human umbilical vein endothelial cells. Data are means ± SE for 4 experiments in duplicate. * P < 0.05 by 1-way ANOVA.

NO production. NO metabolites (nitrate and nitrite) in the conditioned medium accumulated under the static condition, and the level at 90 min was 46 ± 14 pmol/µg protein (Fig. 6). Exposure of the cells to 15 dyn/cm2 laminar flow significantly enhanced the time-dependent increase in NO metabolite concentration in the medium, and the level after 90 min (108 ± 26 pmol/µg protein) was twofold greater than that with a static baseline production (P < 0.05). Further exposure to the same shear (15 dyn/cm2) caused the similar production of NO metabolites (99 ± 16 pmol/µg protein, n = 3). Shear stress at 25 dyn/cm2 enhanced NO metabolite production (194 ± 25 pmol/µg protein) twofold compared with that at 15 dyn/cm2, as was seen for prostacyclin production. Although indomethacin at 10-5 M totally inhibited prostacyclin production not only at baseline (0.3 ± 0.3 pg/µg protein at 90 min) but in a laminar flow condition (2.0 ± 2.0 pg/µg protein), the production of NO metabolites in both static and flow conditions was unaffected.


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  		Fig. 6.   Effect of indomethacin (Indo) on shear (15 dyn/cm2)-induced nitric oxide metabolite (NOx) production in human umbilical vein endothelial cells. Data are means ± SE for 4 experiments in duplicate. * P < 0.05 by 1-way ANOVA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study showed that shear stress at 15 dyn/cm2 increased cumulative production of prostacyclin and NO twofold in HUVEC; that flow-induced prostacyclin production was enhanced by L-NAME and methylene blue, whereas it was attenuated by 8-BrcGMP; that the increase in prostacyclin production induced by GTPgamma S was abolished after treatment with 8-BrcGMP; and that the flow-induced enhancement of NO production was unaffected even after indomethacin totally inhibited prostacyclin production.

Cross talk of prostacyclin and NO production. The enhancement of prostacyclin production by the moderate level of shear stress is consistent with known flow-induced responses in endothelial cells (11). Continuous laminar shear stress in the present study caused a rapid and marked increase in prostacyclin production, which was followed by a sustained phase. Similarly, a burst of NO production also was reported to occur in endothelial cells immediately after the initiation of shear stress. Although the production of prostacyclin and NO was not saturated under the moderate level of shear stress at 15 dyn/cm2, this shear is equal to the level in a physiological condition. Thus we chose the moderate level of shear stress rather than the high level, which may elicit the maximal release of these compounds.

NO was shown to increase intracellular cGMP via the activation of soluble guanylyl cyclase in an autocrine or paracrine fashion (29). To elucidate cross talk of prostacyclin and NO production in endothelial cells, we investigated the effect of the inhibition of NO synthesis on the shear-induced prostacyclin production. The result showed that inhibition of NO synthase with L-NAME enhanced flow-induced production of prostacyclin, indicating that endogenous NO functions as an inhibitor of prostacyclin production in an autocrine or paracrine fashion. Matthews et al. (22) demonstrated that bradykinin-induced prostacyclin production is suppressed by both acute exposure to sodium nitroprusside, the nitrovasodilator drug, and chronic exposure to 3-morpholinosydnonimine, whereas it is enhanced after the inhibition of NO synthase. Barker et al. (2) showed that the release of prostacyclin in response to angiotensin II is enhanced in isolated rings of human saphenous vein when synthesis of NO is inhibited and that nitroglycerin inhibited the angiotensin II-induced release of endogenous prostacyclin. Beverelli et al. (4) reported that chronic inhibition of NO synthase enhances the production of prostacyclin in coronary arteries through upregulation of the cyclooxygenase type 1 isoform. In all, NO may exert a suppressant effect on the prostacyclin production under static conditions. As to the in vivo role of this suppressant effect, clinical studies showed that the NO production is impaired in patients with atherosclerosis (16), whereas the prostacyclin production is not reduced (10). The possibility arises that vessel homeostasis may be maintained through a compensatory increase in prostacyclin in vivo when the output of NO is decreased. The present finding that prostacyclin production is enhanced when NO production is suppressed in vitro may be consistent with this possibility. In contrast to prostacyclin, the cumulative production of NO under shear stress at 15 dyn/cm2 was unaffected even after the complete inhibition of cyclooxygenase. Because prostacyclin receptor is not present in vascular endothelial cells (27), it is reasonable to presume that prostacyclin under flow has no effect on NO production in these cells.

Mechanism for suppressant effect of NO on prostacyclin production. The suppressant effect of NO may be associated with cell death because of its cytotoxic action. However, our results showed no significant reduction in protein content of the dishes. In addition, trypan blue studies demonstrated no loss of viability in HUVEC. It is therefore proposed that mechanisms other than the cytotoxic action are responsible for the suppressant effect of NO. Thus we investigated a role of cGMP, the second messenger of NO, in the reduction in prostacyclin production. Because there is no direct approach to determine the role of cGMP, we addressed this issue with two different approaches using methylene blue, a guanylyl cyclase inhibitor, and 8-BrcGMP, an exogenous cGMP. The results demonstrated that pretreatment with methylene blue enhanced flow-induced prostacyclin production to a similar degree as L-NAME. Although methylene blue has been used extensively as an inhibitor of soluble guanylyl cyclase, it also acts as a direct inhibitor of NO synthase (23). With these cautions, we observed in the present study that exogenous 8-BrcGMP suppressed the flow-induced prostacyclin production in HUVEC. Thus the suppressant effect of methylene blue is likely to be caused by decreased cGMP rather than by diminished NO production. Overall, our data suggest that the effect of NO on prostacyclin production may be dependent on cGMP.

The release of prostacyclin from sheared endothelial cells requires the activation of GTP-binding proteins. Although GTP-binding proteins are only involved in NO production at early time points of shear, the release of prostacyclin is entirely dependent on the activation of GTP-binding proteins during shear. We therefore investigated the mechanism for the suppressant effect of cGMP by reconstructing the condition of activated GTP-binding proteins. Although GTPgamma S, an activator of GTP-binding proteins, is known to have a low permeability in intact cells, the condition used was shown to result in an intracellular concentration of this compound high enough to be effective (7). The enhanced production of prostacyclin by GTPgamma S was abolished after cotreatment with 8-BrcGMP, whereas the enhanced production with ionomycin was unaffected. This suggests the possibility that one site of the action of cGMP may be GTP-binding proteins.

Limitations. The mechanism for the suppressant effect of NO on prostacyclin production under shear stress was investigated by using a number of probes. However, because the effects of these probes are not necessarily specific, we could not reach a conclusion as to the molecular mechanism for the cross talk of prostacyclin and NO. Further investigation will be required to elucidate its mechanism in a shear condition and the in vivo benefit of compensatory increase in prostacyclin when the output of NO is decreased.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. Osanai, Second Dept. of Internal Medicine, Hirosaki Univ. School of Medicine, Hirosaki 036-8562, Japan.

Received 19 January 1999; accepted in final form 10 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 278(1):H233-H238
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