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Prostaglandin synthesis by vascular smooth muscle cells is stimulated by bradykinin, prazosin and hydralazine
PROSTAGLANDIN S Y N T H E S I S BY V A S C U L A R S M O O T H M U S C L E C E L L S IS S T I M U L A T E D BY B R A D Y K I N I N , P R A Z O S I N A N D HYDRALAZINE R. D. DYER,J. J. HUTTNER,S. Y. TAN and P. J. MULROW Department of Medicine, Medical College of Ohio, Toledo, Ohio 43699, U.S.A.
INTRODUCTION
Exogenous prostaglandins (PGs) can change vascular tone 9'~° and PGs have been implicated as intermediates in the vasodilation induced by bradykinin ~2 and infused nicotinic acid. 4 The production of PGE~ by vascular smooth-muscle cells was studied to gain insight into the roles of prostaglandins in the control of vascular tone. MATERIALS
AND
METHODS
Smooth muscle cell cultures were prepared as described 7 from collagenase-digested guinea pig aortas. Forty-eight flasks (25 cm 2) confluent at passage 4 were available for study within 20 days fo the initial culture preparation. For evaluation of PG production, media (Eagles MEM with 25 mM HEPES and 5~ fetal bovine serum) was decanted from the flasks and replaced by 4 ml fresh media. Vasoactive compounds dissolved in serumfree media were added and the cells were incubated at 37°C in a 5~ CO2 atmosphere. At the end of the incubation, media was-decanted and frozen at -80'~C until assay for immuno-PGE2 (iPGE2) as described. 11
Rea#ents (5,6,8,11,12,14,15-3H)PGEa (160 Ci/mmole) was purchased from Amersham (Arlington Heights, IL) and purified by HPLC prior to use. Chromatography solvents were from Burdick and Jackson (Muskegon, MI). Bradykinin, cortisol, cyclandelate, nicotinic acid and papaverine HCI were from Sigma (St. Louis, MO). PGE2 was provided by UpJohn (Kalamazoo, MI), hydralazine by Ciba-Geigy (Summit, N J), prazosin from Pfizer (Brooklyn, NY), diazoxide from Schering (Bloomfield, N J), sodium nitroprusside and nicotinyl alcohol from Hoffmann-LaRoche (Nutley, N J), isoxsuprine from Mead Johnson (Evansville, IN.) and indomethacin from Merk Sharp and Dohme (Rahway, N J). Other chemicals were reagent grade or better and from various commercial suppliers. RESULTS
AND
DISCUSSION
Typical confluent cultures produced 4.5 ___0.3 ng (x + sere; n = 48) iPGE2 in 90 min which is in agreement with reports for similar cultures? iPGE2 production in the absence of fetal bovine serum was not detectable (less than 100 pg/flask), a stimulatory effect of serum which has been noted in cultures of rat medullary cells. -a For cultured cells preincubated with 3 x 10-5 M indomethacin for 30min, the production of iPGE2 was reduced from a control value of 2.7 _ 0.15 to 0.12 _ 0.01 ng! flask/60min. Thus, as with other cell types, t'5'13 the accumulation of iPGE2 in the culture media represents synthesis rather than release of previously synthesized materials. Preincubation of cells with 10-~M cortisol overnight reduced iPGE~ production from 2.7 + 0.15 to 1.5 ___0.01 ng/flask/60min. Inhibitory effects of glucocorticoids on PGE~ production in minced renal papillae 2,1'~ are reported to be dependent on RNA and 557
R D. D.~er et ul.
.'iSg I
STIMULATION OF PGE 2 PROOUCTION BY BRADYKININ
c
o ¢-
l
i c) 0 •
o
I t;-,
1 ,'0-, ,b-,
,b-'
BRADYKININ (Moles/bier)
Fso. I. Brad)ksnin stimulation ol" ~PGE~ pro
protein synthesis and, in transformed mouse fibroblasts, 6 on reduced phospholipase activity. Our results suggest that glucocorticoids may also have a specific action on vascular smooth muscle cells which reduces PGE2 production. iPGE2 production by cultures of aorta smooth muscle cells is sensitive to successive media changes, increasing from 3.04 + 0.23 ng/flask/9Omin in the initial incubation period to 4.63 _+ 0.32 in the second incubation period and then returning to control levels in subsequent incubations. This mild stimulation by media change is much less than the 5 IO-fold increase observed with cultured renal medullary cells. ~ Because iPGE2 production changes in response to media changes, effects of various stimulators on iPGE~ production are compared to control data from flasks which have had the same number of media changes, rather than control data from the same flasks during another incubation period in the absence of the stimulator. Bradykinin causes increased PGE production in a variety of tissues '~'~2''a through the stimulation of phospholipase. As shown in Fig. I. cultured guinea pig smooth muscle cells also respond in a dose-dependent manner to bradykinin. The half-maximal increase in PGE~ synthesis occurs at approximately 10-'~ M bradykinin which is similar to the dose giving half-maximal response for cultured human umbilical vein smooth muscle cells. ~ The apparent decreases in iPGE: production at the lower doses of bradykinin are not significant (P > 0.10}. To evaluate the time course of bradykinin stimulation of iPGE~ synthesis, cells were incubated with 10-SM bradykinin for various times from 10rain to 4.Shr. iPGE~ in control flasks increased with time to a concentration of 13.8 ng/flask at 4.5 hr. As shown in Fig. 2. the time course of bradykinin stimulation exhibits minimal response during the first 30 min of incubation, followed by a dramatic increase in iPGEz production, resulting in a maximal increase above control values at three hours incubation. The effects of 10-" and 10- s M vasoactive compounds on iPGE~ production were evaluated using 3 hr incubation periods. Prazosin HCI and hydralazine HCI caused significant increases in iPGE2 production (Fig. 3}, amounting to 48~o and 68/0, oj respectively, of that achieved with I0- s M bradykinin. Lower doses of the drugs did not increase PGE production significantly. Other drugs tested, including nitroprusside, diazoxide, nicotine acid, nicotinyl alcohol tartate, papaverine hydrochloride, isoxsuprine, and cyclandelatc, had no significant effect on iPGE~ production under these conditions.
Stimulation of P G synthesis by vascular smooth muscle cells
559
TIME COURSE OF BRADYKININ STIMULATION OF PGE 2 SYNTHESIS •
6"
o O
O
E
z < u.I (l: o ~_
2-
e I
o
i INCUBATION TIME (hr) (10-s M Bradykinin)
FIG. 2. Time course of bradykinin stimulation of iPGE2 production. Media was collected from flasks at various times after addition of 10 - s M bradykinin. The increase in iPGEa due to bradykinin was calculated by subtracting the average value for duplicate control flasks (no bradykinin) from the average value for duplicate treatment flasks. Data are from two cell lines.
These results suggest a possible role for prazosin and hydralazine in the stimulation of prostaglandin biosynthesis by vascular smooth-muscle cells. The lack of stimulation of iPGE2 biosynthesis by nicotinic acid may indicate that the indomethacin-blocked vasodilation induced by infused nicotinic acid '~ is due to prostaglandin production by cells other than vascular smooth muscles. These studies illustrate the utility of cultured aorta smooth-muscle cells for the study of the effects of vasoactive agents on prostaglandin production. The cells produce measurable amounts of iPGE2 and exhibit a dose-response relationship to stimulation by bradykinin, a vasodilator known to stimulate prostaglandin production by intact blood vessels, iPGE~synthesis is inhibited by indomethacin and, to a lesser degree, by cortisol. Finally, the synthesis of PGs by vascular smooth muscle cells is increased by the direct-acting vasodilator, hydralazine, and the ~-adrenoceptor blocking agent, prazosin, suggesting a possible role for these drugs in the stimulation of PG biosynthesis by vascular smooth muscle.
22-
EFFECTS OF BRADYKININ AND VASODILATORS ON PROSTAGLANDIN PRODUCTION
~o 18 03
|
_~ .~
u~ ~4 ........ { ...............................................................
n
} ........
lO
Con;rol
B'K
PR~AZ
HY~)R
DI~Z
FIG. 3. Stimulation of PGEz production by vasoactive compounds. Confluent cells were incu-
bated with 10-SM bradykinin (BK), prazosin (PRAZ), hydralazine (HYDR) and diazoxide (DIAZ) for 3 hr and PGEe measured in the media by RIA. Typical data from one cell line, means and standard errors from duplicate flasks.
560
R.D. Dyer et al. SUMMARY
The effects of vasodilators on the synthesis of prostaglandins by cultured vascular smooth muscle cells were studied to gain insight into the roles of prostaglandins in the control of vascular tone. Cells from collagenase-digested guinea pig aorta were grown in media containing 5 ~ fetal bovine serum and used at confluency in 25cm 2 flasks. Immuno-PGE2 (iPGE2) in the culture medium was determined by RIA. Typical cultures produced 4.5 + 0.3 (SE)ng iPGE2 in 90 min. Preincubation of the cells with 30/~M indomethacin or 10/~M cortisol reduced iPGE2 synthesis by 95~ and 44Yo, respectively. iPGE2 synthesis was stimulated in a dose-response fashion by bradykinin(BK) with half-maximal stimulation at approximately 10-7 M. BK stimulated iPGE2 synthesis was maximal at 3 hr incubation time. Prazosin HCI and hydralazine HC1, when incubated with the cells for 3 hr at 10- 5 M, elicited a stimulation of iPGE2 synthesis which was 48~ and 68~, respectively, of that achieved with 10-5 M BK. Other vasodilators, including nitroprusside, nicotinic acid, and diazoxide, had no measurable effect on iPGE2 production at concentrations up to 10 -5 M. These results suggest a possible role for prazosin and hydralazine in the stimulation of prostaglandin biodynthesis in smooth muscle cells. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I. 12: 13. 14.
ALEXANDER,R. W. and GIMBRONE,JR., M. A. Proc. Nat. Acad. Sci. USA 75, 1617-1670 (1976). DaNO~, A. and ASSOULINE,G. Nature 273, 552-554 (1978). DUNN, M. J., STaLEY, R. S. and HARRISON,M. Prostaglandins 12, 37-49 (1976). EKLUND,B., KAIJSER,L., NOWaK, J. and WENNMALM,AKE. Prostaglandins 17, 821-830 (1979). Ho~G, S. L. and LEWNE,L. J. biol. Chem. 251, 5814-5816 (1976). HONG, S. L. and LEVI~qE,L. Proc. Natl. Acad. Sci. USA 73, 1730-1734 (1976). HUTTNER,J. J.. CORNWELL,D. D. and MILO, G. E. TCA Manual 3, 633-636 (1977). HUTTNER,J. J., GWEBU, E. T., PANGANAMALA,R. V., MILO, G. E., CORNWELL,D. G., SHARMA,H. M. and GEER, J. C. Science 197, 289-291 (1977). ROBINSON,B. F., COLLIER,J. G., KARIM,S. M. M. and SOMERS,K. Clin. Sci. 44, 367-376 (1973). SZCZEKLIK,A. and GRYGLEWSKI,R. J. In Prostacyclin, pp. 393-407. (VANE, J. R. and BERGSTROM,S., eds.) Raven Press. New York. 1979. TAN, S. Y., SaNI~WlSCH,D. W. and MULROW, P. J. Prostaglandins Med. 4, 53-63 (1980). TERRAGANO,D. A., CROWSHAW, K., TERRAGNO, N. A. and MCGIFF, J. C. Circ. Res. 36-37, Suppl. 1, 1-76-I-80 (1975). ZUSMAN,R. M. and KEISER,H. R. J. biol. Chem. 252, 2069-2071 (1977). ZUSMAN,R. M. and KEISER,H. R. J. clin. ln~,est. 60, 215-223 (1977).
INTRODUCTION
Exogenous prostaglandins (PGs) can change vascular tone 9'~° and PGs have been implicated as intermediates in the vasodilation induced by bradykinin ~2 and infused nicotinic acid. 4 The production of PGE~ by vascular smooth-muscle cells was studied to gain insight into the roles of prostaglandins in the control of vascular tone. MATERIALS
AND
METHODS
Smooth muscle cell cultures were prepared as described 7 from collagenase-digested guinea pig aortas. Forty-eight flasks (25 cm 2) confluent at passage 4 were available for study within 20 days fo the initial culture preparation. For evaluation of PG production, media (Eagles MEM with 25 mM HEPES and 5~ fetal bovine serum) was decanted from the flasks and replaced by 4 ml fresh media. Vasoactive compounds dissolved in serumfree media were added and the cells were incubated at 37°C in a 5~ CO2 atmosphere. At the end of the incubation, media was-decanted and frozen at -80'~C until assay for immuno-PGE2 (iPGE2) as described. 11
Rea#ents (5,6,8,11,12,14,15-3H)PGEa (160 Ci/mmole) was purchased from Amersham (Arlington Heights, IL) and purified by HPLC prior to use. Chromatography solvents were from Burdick and Jackson (Muskegon, MI). Bradykinin, cortisol, cyclandelate, nicotinic acid and papaverine HCI were from Sigma (St. Louis, MO). PGE2 was provided by UpJohn (Kalamazoo, MI), hydralazine by Ciba-Geigy (Summit, N J), prazosin from Pfizer (Brooklyn, NY), diazoxide from Schering (Bloomfield, N J), sodium nitroprusside and nicotinyl alcohol from Hoffmann-LaRoche (Nutley, N J), isoxsuprine from Mead Johnson (Evansville, IN.) and indomethacin from Merk Sharp and Dohme (Rahway, N J). Other chemicals were reagent grade or better and from various commercial suppliers. RESULTS
AND
DISCUSSION
Typical confluent cultures produced 4.5 ___0.3 ng (x + sere; n = 48) iPGE2 in 90 min which is in agreement with reports for similar cultures? iPGE2 production in the absence of fetal bovine serum was not detectable (less than 100 pg/flask), a stimulatory effect of serum which has been noted in cultures of rat medullary cells. -a For cultured cells preincubated with 3 x 10-5 M indomethacin for 30min, the production of iPGE2 was reduced from a control value of 2.7 _ 0.15 to 0.12 _ 0.01 ng! flask/60min. Thus, as with other cell types, t'5'13 the accumulation of iPGE2 in the culture media represents synthesis rather than release of previously synthesized materials. Preincubation of cells with 10-~M cortisol overnight reduced iPGE~ production from 2.7 + 0.15 to 1.5 ___0.01 ng/flask/60min. Inhibitory effects of glucocorticoids on PGE~ production in minced renal papillae 2,1'~ are reported to be dependent on RNA and 557
R D. D.~er et ul.
.'iSg I
STIMULATION OF PGE 2 PROOUCTION BY BRADYKININ
c
o ¢-
l
i c) 0 •
o
I t;-,
1 ,'0-, ,b-,
,b-'
BRADYKININ (Moles/bier)
Fso. I. Brad)ksnin stimulation ol" ~PGE~ pro
protein synthesis and, in transformed mouse fibroblasts, 6 on reduced phospholipase activity. Our results suggest that glucocorticoids may also have a specific action on vascular smooth muscle cells which reduces PGE2 production. iPGE2 production by cultures of aorta smooth muscle cells is sensitive to successive media changes, increasing from 3.04 + 0.23 ng/flask/9Omin in the initial incubation period to 4.63 _+ 0.32 in the second incubation period and then returning to control levels in subsequent incubations. This mild stimulation by media change is much less than the 5 IO-fold increase observed with cultured renal medullary cells. ~ Because iPGE2 production changes in response to media changes, effects of various stimulators on iPGE~ production are compared to control data from flasks which have had the same number of media changes, rather than control data from the same flasks during another incubation period in the absence of the stimulator. Bradykinin causes increased PGE production in a variety of tissues '~'~2''a through the stimulation of phospholipase. As shown in Fig. I. cultured guinea pig smooth muscle cells also respond in a dose-dependent manner to bradykinin. The half-maximal increase in PGE~ synthesis occurs at approximately 10-'~ M bradykinin which is similar to the dose giving half-maximal response for cultured human umbilical vein smooth muscle cells. ~ The apparent decreases in iPGE: production at the lower doses of bradykinin are not significant (P > 0.10}. To evaluate the time course of bradykinin stimulation of iPGE~ synthesis, cells were incubated with 10-SM bradykinin for various times from 10rain to 4.Shr. iPGE~ in control flasks increased with time to a concentration of 13.8 ng/flask at 4.5 hr. As shown in Fig. 2. the time course of bradykinin stimulation exhibits minimal response during the first 30 min of incubation, followed by a dramatic increase in iPGEz production, resulting in a maximal increase above control values at three hours incubation. The effects of 10-" and 10- s M vasoactive compounds on iPGE~ production were evaluated using 3 hr incubation periods. Prazosin HCI and hydralazine HCI caused significant increases in iPGE2 production (Fig. 3}, amounting to 48~o and 68/0, oj respectively, of that achieved with I0- s M bradykinin. Lower doses of the drugs did not increase PGE production significantly. Other drugs tested, including nitroprusside, diazoxide, nicotine acid, nicotinyl alcohol tartate, papaverine hydrochloride, isoxsuprine, and cyclandelatc, had no significant effect on iPGE~ production under these conditions.
Stimulation of P G synthesis by vascular smooth muscle cells
559
TIME COURSE OF BRADYKININ STIMULATION OF PGE 2 SYNTHESIS •
6"
o O
O
E
z < u.I (l: o ~_
2-
e I
o
i INCUBATION TIME (hr) (10-s M Bradykinin)
FIG. 2. Time course of bradykinin stimulation of iPGE2 production. Media was collected from flasks at various times after addition of 10 - s M bradykinin. The increase in iPGEa due to bradykinin was calculated by subtracting the average value for duplicate control flasks (no bradykinin) from the average value for duplicate treatment flasks. Data are from two cell lines.
These results suggest a possible role for prazosin and hydralazine in the stimulation of prostaglandin biosynthesis by vascular smooth-muscle cells. The lack of stimulation of iPGE2 biosynthesis by nicotinic acid may indicate that the indomethacin-blocked vasodilation induced by infused nicotinic acid '~ is due to prostaglandin production by cells other than vascular smooth muscles. These studies illustrate the utility of cultured aorta smooth-muscle cells for the study of the effects of vasoactive agents on prostaglandin production. The cells produce measurable amounts of iPGE2 and exhibit a dose-response relationship to stimulation by bradykinin, a vasodilator known to stimulate prostaglandin production by intact blood vessels, iPGE~synthesis is inhibited by indomethacin and, to a lesser degree, by cortisol. Finally, the synthesis of PGs by vascular smooth muscle cells is increased by the direct-acting vasodilator, hydralazine, and the ~-adrenoceptor blocking agent, prazosin, suggesting a possible role for these drugs in the stimulation of PG biosynthesis by vascular smooth muscle.
22-
EFFECTS OF BRADYKININ AND VASODILATORS ON PROSTAGLANDIN PRODUCTION
~o 18 03
|
_~ .~
u~ ~4 ........ { ...............................................................
n
} ........
lO
Con;rol
B'K
PR~AZ
HY~)R
DI~Z
FIG. 3. Stimulation of PGEz production by vasoactive compounds. Confluent cells were incu-
bated with 10-SM bradykinin (BK), prazosin (PRAZ), hydralazine (HYDR) and diazoxide (DIAZ) for 3 hr and PGEe measured in the media by RIA. Typical data from one cell line, means and standard errors from duplicate flasks.
560
R.D. Dyer et al. SUMMARY
The effects of vasodilators on the synthesis of prostaglandins by cultured vascular smooth muscle cells were studied to gain insight into the roles of prostaglandins in the control of vascular tone. Cells from collagenase-digested guinea pig aorta were grown in media containing 5 ~ fetal bovine serum and used at confluency in 25cm 2 flasks. Immuno-PGE2 (iPGE2) in the culture medium was determined by RIA. Typical cultures produced 4.5 + 0.3 (SE)ng iPGE2 in 90 min. Preincubation of the cells with 30/~M indomethacin or 10/~M cortisol reduced iPGE2 synthesis by 95~ and 44Yo, respectively. iPGE2 synthesis was stimulated in a dose-response fashion by bradykinin(BK) with half-maximal stimulation at approximately 10-7 M. BK stimulated iPGE2 synthesis was maximal at 3 hr incubation time. Prazosin HCI and hydralazine HC1, when incubated with the cells for 3 hr at 10- 5 M, elicited a stimulation of iPGE2 synthesis which was 48~ and 68~, respectively, of that achieved with 10-5 M BK. Other vasodilators, including nitroprusside, nicotinic acid, and diazoxide, had no measurable effect on iPGE2 production at concentrations up to 10 -5 M. These results suggest a possible role for prazosin and hydralazine in the stimulation of prostaglandin biodynthesis in smooth muscle cells. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I. 12: 13. 14.
ALEXANDER,R. W. and GIMBRONE,JR., M. A. Proc. Nat. Acad. Sci. USA 75, 1617-1670 (1976). DaNO~, A. and ASSOULINE,G. Nature 273, 552-554 (1978). DUNN, M. J., STaLEY, R. S. and HARRISON,M. Prostaglandins 12, 37-49 (1976). EKLUND,B., KAIJSER,L., NOWaK, J. and WENNMALM,AKE. Prostaglandins 17, 821-830 (1979). Ho~G, S. L. and LEWNE,L. J. biol. Chem. 251, 5814-5816 (1976). HONG, S. L. and LEVI~qE,L. Proc. Natl. Acad. Sci. USA 73, 1730-1734 (1976). HUTTNER,J. J.. CORNWELL,D. D. and MILO, G. E. TCA Manual 3, 633-636 (1977). HUTTNER,J. J., GWEBU, E. T., PANGANAMALA,R. V., MILO, G. E., CORNWELL,D. G., SHARMA,H. M. and GEER, J. C. Science 197, 289-291 (1977). ROBINSON,B. F., COLLIER,J. G., KARIM,S. M. M. and SOMERS,K. Clin. Sci. 44, 367-376 (1973). SZCZEKLIK,A. and GRYGLEWSKI,R. J. In Prostacyclin, pp. 393-407. (VANE, J. R. and BERGSTROM,S., eds.) Raven Press. New York. 1979. TAN, S. Y., SaNI~WlSCH,D. W. and MULROW, P. J. Prostaglandins Med. 4, 53-63 (1980). TERRAGANO,D. A., CROWSHAW, K., TERRAGNO, N. A. and MCGIFF, J. C. Circ. Res. 36-37, Suppl. 1, 1-76-I-80 (1975). ZUSMAN,R. M. and KEISER,H. R. J. biol. Chem. 252, 2069-2071 (1977). ZUSMAN,R. M. and KEISER,H. R. J. clin. ln~,est. 60, 215-223 (1977).