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Effects of calcium and parathyroid hormone on prostacyclin synthesis by vascular tissue
I.ife S c i e n c e s , Vol. 40, P r i n t e d in t h e U . S . A .
pp.
983-986
Pergamon Journal~
EFFECTS OF CALCIUM AND P A R A T H Y R O I D HORMONE ON PROSTACYCLIN SYNTHESlS BY VASCULAR TISSUE P. Lopez-Jaramillo,
F. G u a r n e r and S. Moncada
The Wellcome Research Laboratories Langley C o u r t , Beckenham, Kent BR3 3BS, U . K . (Received
in
final
f o r m December 4,
1986)
Summary P r o s t a c y c l i n g e n e r a t i o n by rat aortic r i n g s was studied at d i f f e r e n t calcium concentrations. Extracellular calcium influenced prostacyclin synthesis, as reflected by the release of 6-keto-PGF_ into the medium, in a concentration-dependent fashion. C1~cium levels beyond the physiological range (1.12-1.25 mM u n b o u n d calcium) markedly stimulated 6-keto-PGF. production when compared w i t h calcmm-free s o l u t i o n s . On the o t h e r h a n d , addition of purified parathyroid hormone did not change 6-keto-PGF. p r o d u c t i o n at any calcium c o n c e n t r a t i o n tested. These data1~suggest that p a r a t h y r o i d hormone has no d i r e c t effect on p r o s t a c y c l i n s y n t h e s i s by vascular tissue, a l t h o u g h it might i n f l u e n c e p r o s t a c y c l i n g e n e r a t i o n t h r o u g h changes in e x t r a c e l l u l a r calcium levels. •
.
io.
T h e f i r s t step in the generation of eicosanoids is the appearance of free a r a c h i d o n i c acid l i h e r a t e d by the action of phospholipases. A l t h o u g h it is well known that the presence of calcium is essential for the c a t a l y t i c a c t i v i t y of the phospholipases (1), w h e t h e r i n t r a c e l l u l a r or e x t r a c e l l u l a r calcium is involved in this process remains controversial (2,3). Nevertheless, stimulation of endothelial p r o s t a c y c l i n s y n t h e s i s b y b r a d y k i n i n (4), calcium ionophore (5), the thromboxane A_ mimetic U-46619 (6) or t h r o m b i n (3) seems to be mediated by influx of extracellular calcium in a c a l m o d u l i n - d e p e n d e n t process ( 7 ) . On the o t h e r h a n d , calcium plays a c r i t i c a l role in v a s c u l a r smooth muscle f u n c t i o n ( 8 ) . While its presence is essential for c o n t r a c t i l i t y , a v a s o r e l a x i n g action of calcium has also been documented ( 9 , 1 0 ) . Thus, increases in e x t r a c e l l u l a r calcium are associated w i t h vascular r e l a x a t i o n and vasodilation (10). P a r a t h y r o i d hormone (PTH) increases c i r c u l a t i n g levels of calcium and, interestingly, parathyroid extracts, purified parathyroid hormone and s y n t h e t i c PTH 1-34 have been shown to induce vascular relaxation (11,12). Its vasoclilator effect on c o r o n a r y a r t e r i e s is not i n f l u e n c e d b y a d r e n e r g i c , c h o l i n e r g i c or h i s t a m i n e r g i c agonists or antagonists (13). Since p r o s t a c y c l i n is a potent v a s o d i l a t o r (14), the aim of t h i s s t u d y was to investigate whether extracellular calcium and PTH stimulate p r o s t a c y c l i n b i o s y n t h e s i s by v a s c u l a r tissue. Copyright
0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals l,td.
984
Effect
of Calcium
on Prostacyclin
Synthesis
Vol.
40, No.
10,
L987
Methods Male Wistar rats (180-250 g body weight) were sacrificed by cervical dislocation and the abdominal aorta was immediately removed and cut into ri n g s as described elsewhere (15). Five aortic r i n g s , about 0.7 mg wet weight each, were pooled in a test tube with I ml calcium-free Krebs' b u f f e r c o n t a i n i n g 0.5-~ bovine serum albumin p r e v i o u s l y gassed to pH 7.4 with 95 ° O_ and 5go C O m . A f t e r two hours i n c u b a t i o n at 37°C, the medium was reZmoved and r i n g s washed twice with ice-cold b u f f e r . " E x h a u s t e d " rings were then incubated in 500 lal a l b u m i n - f r e e Krebs' for a f u r t h e r 30 minutes with d i f f e r e n t c o n c e n t r a t i o n s o f calcium (added as calcium c h l o r i d e ) or p u r i f i e d bovine PTH (Sigma, St. Louis, M o . ) . F i n a l l y , s u p e r n a t a n t s were collected and stored with 10 p g l m l indomethacin (Sigma) at 2 0 ° C . Prostacyclin release was estimated by radio-immunoassay of the stable d e r i v a t i v e 6-keto-PGF1cx, as p r e v i o u s l y d es c r ibed (16). Results are shown as the mean _+ s t a n d a r d e r r o r of f i v e incubations per p o i n t . Statistical d i f f e r e n c e s were analysed by the Student's t - t e s t . Results E x t r a c e l l u l a r calcium s i g n i f i c a n t l y influenced 6-keto-PGF.] g e n e r a t i o n by vascular tissue in a c o n c e n t r a t i o n - d e p e n d e n t manner. ConcelnC~rations beyond the p h y s i o l o g i c a l range (in p r o t e i n - f r e e media the physiological range is considered to be 1.12-1.25 raM) m a r k e d l y increased 6-keto-PGF1c~ release as compared to c a l c i u m - f r e e solutions (see f i g u r e I ) . 25
t
A e-
E 20
v-Ll.
I
o
15
I
10
I
0
I 0.625
I 1.25
I
I 2.5
Calcium (raM) FIG. I Generation of 6-keto-PGF. by rat a o r t i c r i n g s incubated in Krebs' b u f f e r at dufferent calc,um c onc e n t r a t , o n s . Mean _+ standard e r r o r of five e x p e r i m e n t s are shown. The values at a calcium c o n c e n t r a t i o n of 2.5 mM are s i g n i f i c a n t l y (p < 0.01) h i g h e r than at lower calcium c o n c e n t r a t i o n s . •
]Or.
.
.
Vol. 40, No.
10, 1987
Effect of Calcium on Prostacyclin
Synthesis
985
Purified bovine PTH was studied at d i f f e r e n t calcium concentrations. No effect associated with the presence of PTH could be observed at any level tested, while calcium stimulated 6-keto-PGFl~ production independently of the PTH concentration (see f i g u r e 2). Fih~lly, indomethacin (10 lug/ml) reduced by 90% (n=6) both baseline production and calcium-dependent stimulation of 6-keto-PGF1 .
35
"E
°oo
30
PTH pmol/I
50O 1000
~ 25
I
10,000
15
10 0
0.625
1.25
2.5
Calcium (mM)
FIG. 2 Effects of p a r a t h y r o i d hormone (PTH) on 6-keto-PGF_ production by rat aortic rings incubated in Krebs' b u f f e r at difflearent calcium concentrations. No changes associated with the addition of PTH to the medium were observed while calcium stimulates 6-keto-PGFlo generation independently of the presence of PTH. Discussion The present results show that in v i t r o prostacyclin generation by vascular tissue, as reflected by 6-keto-PGF. , . is dependent on e x t r a c e l l u l a r • . ] calcium levels. Aortic tissue ,ncubated ,n ca°lclum-free medium yielded lower 6-keto-PGF.] ( 3 l production than specimens incubated at physiological calcium levels, but major changes in prostacyclin generation were observed by f u r t h e r increases in calcium concentration beyond the physiological range. Similar f i n d i n g s were r e c e n t l y reported by Brown and Swartz (17) using dispersed cells or fragments from bovine p a r a t h y r o i d glands.
986
Effect
of Calcium
on Prostacvclin
Svnthesis
VoI.
40, N(~. I0,
1987
Previous experimental work r e p o r t e d that the presence of calcium in the medium is essential for the stimulation of p r o s t a c y c l i n s y n t h e s i s by hormones such as n o r a d r e n a l i n e , a n g i o t e n s i n II or b r a d y k i n i n ( 4 , 5 , 7 ) and suggested that an i n f l u x of e x t r a c e l l u l a r calcium would be i n v o l v e d in the process. O u r data s u p p o r t this view in that a h i g h n o n - p h y s i o l o g i c a l calcium concentration, w i t h o u t any f u r t h e r stimulus, is capable of i n c r e a s i n g prostacyclin synthesis. Rises in e x t r a c e l l u l a r calcium would d i r e c t l y induce calcium e n t r y t h r o u g h a c t i v a t i o n of the calmodulin complex ( 7 ) , which is known to stimulate phospholipase A 2 a c t i v i t y (18). P u r i f i e d PTH is known to relax vascular smooth muscle and it has been proposed that p r o s t a g l a n d i n s y n t h e s i s could be i n v o l v e d in this effect, for indomethacin was shown to i n h i b i t t h i s action (19). Our data indicate t h a t , u n l i k e calcium, p u r i f i e d b o v i n e PTH does not d i r e c t l y increase p r o s t a c y c l i n p r o d u c t i o n b y rat aortic r i n g s in v i t r o which makes the suggested mechanism unlikely. However, o u r data also suggest that changes in the e x t r a c e l l u l a r calcium levels mediated by PTH in v i v o may i n f l u e n c e p r o s t a c y c l i n s y n t h e s i s . F u r t h e r e x p e r i m e n t s should e x p l o r e this h y p o t h e s i s . References I. 2. 3 4 5 6 7 8 9 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
H. VAN DEN BOSCH, Biochim. B i o p h y s . Acta. 604 191-246 (1980). B . B . WEKSLER, C.W. LEY and E . A . JAFFE, J. C l i n . I n v e s t . 62 923-930 (1978). A F . A . BROTHERTON and J . C . HOAK, Proc. Natl. Acad. Sci. USA 79 495-499 (1982). D J. C R U T C H L E Y , J.W. RYAN, U . S . RYAN and G . H . FISCHER, Biochim. B i o p h y s . Acta 751, 99-107 (1983). A R. WHORTON, C . E . WILLIS, R.S. KENT and S . L . YOUNG, Lipids 19 17-24 (1984). J Y. JEREMY, D.P. MIKHAILIDIS and P. DANDONA, Eur. J. Pharmacol. 107 259-262 (1985). D STEWART, E. POUNTNEY and D. F I T C H E T T , Can. J. Physiol. Pharmacol. 62 1341-1346 (1984). H K U R I Y A M A , Y. ITO, H. SUZUKI, K. K I T A M U R A and T . ITOH, Am. J. P h y s i o l . 243 H641-H662 (1982). D . F . BOHR, Science 139 597-599 (1963). R . C . WEBB and D . H . BOHR, Am. J. Physiol. 235 C227-C232 (1978). G . A . CHARBON, Eur. J. Pharmacol. 3 275-278 (1968). D . A . MCCARRON, D . H . ELLISON and S. ANDERSON, Am. J. Physiol. 246, F96-F100 (1984). M . F . CRASS and P . K . T . PANG, Science 207 1087-1089 (1980). S. MONCADA and J . R . VANE, Pharmacol. Rev. 30 293-331 (1979). S. MONCADA, E . A . HIGGS and J . R . VANE, Lancet i 18-21 (1977). J . A . SALMON, Prostaglandins 15 383-396 (1978). E.M. BROWN and S . L . SWARTZ, Prostaglandins 29 35-46 (1985). P . Y . K . WONG and W.Y. CHEUNG, Biochem. B i o p h y s . Res. Commun. 90 473-480 (1979). Y. SAGLIKES, S.G. MASSRY, K . , ISEKI, J . L . , NADLER AND V . M . CAMPESE, Am. J. Physiol. 248 F674-F681 (1985).
pp.
983-986
Pergamon Journal~
EFFECTS OF CALCIUM AND P A R A T H Y R O I D HORMONE ON PROSTACYCLIN SYNTHESlS BY VASCULAR TISSUE P. Lopez-Jaramillo,
F. G u a r n e r and S. Moncada
The Wellcome Research Laboratories Langley C o u r t , Beckenham, Kent BR3 3BS, U . K . (Received
in
final
f o r m December 4,
1986)
Summary P r o s t a c y c l i n g e n e r a t i o n by rat aortic r i n g s was studied at d i f f e r e n t calcium concentrations. Extracellular calcium influenced prostacyclin synthesis, as reflected by the release of 6-keto-PGF_ into the medium, in a concentration-dependent fashion. C1~cium levels beyond the physiological range (1.12-1.25 mM u n b o u n d calcium) markedly stimulated 6-keto-PGF. production when compared w i t h calcmm-free s o l u t i o n s . On the o t h e r h a n d , addition of purified parathyroid hormone did not change 6-keto-PGF. p r o d u c t i o n at any calcium c o n c e n t r a t i o n tested. These data1~suggest that p a r a t h y r o i d hormone has no d i r e c t effect on p r o s t a c y c l i n s y n t h e s i s by vascular tissue, a l t h o u g h it might i n f l u e n c e p r o s t a c y c l i n g e n e r a t i o n t h r o u g h changes in e x t r a c e l l u l a r calcium levels. •
.
io.
T h e f i r s t step in the generation of eicosanoids is the appearance of free a r a c h i d o n i c acid l i h e r a t e d by the action of phospholipases. A l t h o u g h it is well known that the presence of calcium is essential for the c a t a l y t i c a c t i v i t y of the phospholipases (1), w h e t h e r i n t r a c e l l u l a r or e x t r a c e l l u l a r calcium is involved in this process remains controversial (2,3). Nevertheless, stimulation of endothelial p r o s t a c y c l i n s y n t h e s i s b y b r a d y k i n i n (4), calcium ionophore (5), the thromboxane A_ mimetic U-46619 (6) or t h r o m b i n (3) seems to be mediated by influx of extracellular calcium in a c a l m o d u l i n - d e p e n d e n t process ( 7 ) . On the o t h e r h a n d , calcium plays a c r i t i c a l role in v a s c u l a r smooth muscle f u n c t i o n ( 8 ) . While its presence is essential for c o n t r a c t i l i t y , a v a s o r e l a x i n g action of calcium has also been documented ( 9 , 1 0 ) . Thus, increases in e x t r a c e l l u l a r calcium are associated w i t h vascular r e l a x a t i o n and vasodilation (10). P a r a t h y r o i d hormone (PTH) increases c i r c u l a t i n g levels of calcium and, interestingly, parathyroid extracts, purified parathyroid hormone and s y n t h e t i c PTH 1-34 have been shown to induce vascular relaxation (11,12). Its vasoclilator effect on c o r o n a r y a r t e r i e s is not i n f l u e n c e d b y a d r e n e r g i c , c h o l i n e r g i c or h i s t a m i n e r g i c agonists or antagonists (13). Since p r o s t a c y c l i n is a potent v a s o d i l a t o r (14), the aim of t h i s s t u d y was to investigate whether extracellular calcium and PTH stimulate p r o s t a c y c l i n b i o s y n t h e s i s by v a s c u l a r tissue. Copyright
0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals l,td.
984
Effect
of Calcium
on Prostacyclin
Synthesis
Vol.
40, No.
10,
L987
Methods Male Wistar rats (180-250 g body weight) were sacrificed by cervical dislocation and the abdominal aorta was immediately removed and cut into ri n g s as described elsewhere (15). Five aortic r i n g s , about 0.7 mg wet weight each, were pooled in a test tube with I ml calcium-free Krebs' b u f f e r c o n t a i n i n g 0.5-~ bovine serum albumin p r e v i o u s l y gassed to pH 7.4 with 95 ° O_ and 5go C O m . A f t e r two hours i n c u b a t i o n at 37°C, the medium was reZmoved and r i n g s washed twice with ice-cold b u f f e r . " E x h a u s t e d " rings were then incubated in 500 lal a l b u m i n - f r e e Krebs' for a f u r t h e r 30 minutes with d i f f e r e n t c o n c e n t r a t i o n s o f calcium (added as calcium c h l o r i d e ) or p u r i f i e d bovine PTH (Sigma, St. Louis, M o . ) . F i n a l l y , s u p e r n a t a n t s were collected and stored with 10 p g l m l indomethacin (Sigma) at 2 0 ° C . Prostacyclin release was estimated by radio-immunoassay of the stable d e r i v a t i v e 6-keto-PGF1cx, as p r e v i o u s l y d es c r ibed (16). Results are shown as the mean _+ s t a n d a r d e r r o r of f i v e incubations per p o i n t . Statistical d i f f e r e n c e s were analysed by the Student's t - t e s t . Results E x t r a c e l l u l a r calcium s i g n i f i c a n t l y influenced 6-keto-PGF.] g e n e r a t i o n by vascular tissue in a c o n c e n t r a t i o n - d e p e n d e n t manner. ConcelnC~rations beyond the p h y s i o l o g i c a l range (in p r o t e i n - f r e e media the physiological range is considered to be 1.12-1.25 raM) m a r k e d l y increased 6-keto-PGF1c~ release as compared to c a l c i u m - f r e e solutions (see f i g u r e I ) . 25
t
A e-
E 20
v-Ll.
I
o
15
I
10
I
0
I 0.625
I 1.25
I
I 2.5
Calcium (raM) FIG. I Generation of 6-keto-PGF. by rat a o r t i c r i n g s incubated in Krebs' b u f f e r at dufferent calc,um c onc e n t r a t , o n s . Mean _+ standard e r r o r of five e x p e r i m e n t s are shown. The values at a calcium c o n c e n t r a t i o n of 2.5 mM are s i g n i f i c a n t l y (p < 0.01) h i g h e r than at lower calcium c o n c e n t r a t i o n s . •
]Or.
.
.
Vol. 40, No.
10, 1987
Effect of Calcium on Prostacyclin
Synthesis
985
Purified bovine PTH was studied at d i f f e r e n t calcium concentrations. No effect associated with the presence of PTH could be observed at any level tested, while calcium stimulated 6-keto-PGFl~ production independently of the PTH concentration (see f i g u r e 2). Fih~lly, indomethacin (10 lug/ml) reduced by 90% (n=6) both baseline production and calcium-dependent stimulation of 6-keto-PGF1 .
35
"E
°oo
30
PTH pmol/I
50O 1000
~ 25
I
10,000
15
10 0
0.625
1.25
2.5
Calcium (mM)
FIG. 2 Effects of p a r a t h y r o i d hormone (PTH) on 6-keto-PGF_ production by rat aortic rings incubated in Krebs' b u f f e r at difflearent calcium concentrations. No changes associated with the addition of PTH to the medium were observed while calcium stimulates 6-keto-PGFlo generation independently of the presence of PTH. Discussion The present results show that in v i t r o prostacyclin generation by vascular tissue, as reflected by 6-keto-PGF. , . is dependent on e x t r a c e l l u l a r • . ] calcium levels. Aortic tissue ,ncubated ,n ca°lclum-free medium yielded lower 6-keto-PGF.] ( 3 l production than specimens incubated at physiological calcium levels, but major changes in prostacyclin generation were observed by f u r t h e r increases in calcium concentration beyond the physiological range. Similar f i n d i n g s were r e c e n t l y reported by Brown and Swartz (17) using dispersed cells or fragments from bovine p a r a t h y r o i d glands.
986
Effect
of Calcium
on Prostacvclin
Svnthesis
VoI.
40, N(~. I0,
1987
Previous experimental work r e p o r t e d that the presence of calcium in the medium is essential for the stimulation of p r o s t a c y c l i n s y n t h e s i s by hormones such as n o r a d r e n a l i n e , a n g i o t e n s i n II or b r a d y k i n i n ( 4 , 5 , 7 ) and suggested that an i n f l u x of e x t r a c e l l u l a r calcium would be i n v o l v e d in the process. O u r data s u p p o r t this view in that a h i g h n o n - p h y s i o l o g i c a l calcium concentration, w i t h o u t any f u r t h e r stimulus, is capable of i n c r e a s i n g prostacyclin synthesis. Rises in e x t r a c e l l u l a r calcium would d i r e c t l y induce calcium e n t r y t h r o u g h a c t i v a t i o n of the calmodulin complex ( 7 ) , which is known to stimulate phospholipase A 2 a c t i v i t y (18). P u r i f i e d PTH is known to relax vascular smooth muscle and it has been proposed that p r o s t a g l a n d i n s y n t h e s i s could be i n v o l v e d in this effect, for indomethacin was shown to i n h i b i t t h i s action (19). Our data indicate t h a t , u n l i k e calcium, p u r i f i e d b o v i n e PTH does not d i r e c t l y increase p r o s t a c y c l i n p r o d u c t i o n b y rat aortic r i n g s in v i t r o which makes the suggested mechanism unlikely. However, o u r data also suggest that changes in the e x t r a c e l l u l a r calcium levels mediated by PTH in v i v o may i n f l u e n c e p r o s t a c y c l i n s y n t h e s i s . F u r t h e r e x p e r i m e n t s should e x p l o r e this h y p o t h e s i s . References I. 2. 3 4 5 6 7 8 9 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
H. VAN DEN BOSCH, Biochim. B i o p h y s . Acta. 604 191-246 (1980). B . B . WEKSLER, C.W. LEY and E . A . JAFFE, J. C l i n . I n v e s t . 62 923-930 (1978). A F . A . BROTHERTON and J . C . HOAK, Proc. Natl. Acad. Sci. USA 79 495-499 (1982). D J. C R U T C H L E Y , J.W. RYAN, U . S . RYAN and G . H . FISCHER, Biochim. B i o p h y s . Acta 751, 99-107 (1983). A R. WHORTON, C . E . WILLIS, R.S. KENT and S . L . YOUNG, Lipids 19 17-24 (1984). J Y. JEREMY, D.P. MIKHAILIDIS and P. DANDONA, Eur. J. Pharmacol. 107 259-262 (1985). D STEWART, E. POUNTNEY and D. F I T C H E T T , Can. J. Physiol. Pharmacol. 62 1341-1346 (1984). H K U R I Y A M A , Y. ITO, H. SUZUKI, K. K I T A M U R A and T . ITOH, Am. J. P h y s i o l . 243 H641-H662 (1982). D . F . BOHR, Science 139 597-599 (1963). R . C . WEBB and D . H . BOHR, Am. J. Physiol. 235 C227-C232 (1978). G . A . CHARBON, Eur. J. Pharmacol. 3 275-278 (1968). D . A . MCCARRON, D . H . ELLISON and S. ANDERSON, Am. J. Physiol. 246, F96-F100 (1984). M . F . CRASS and P . K . T . PANG, Science 207 1087-1089 (1980). S. MONCADA and J . R . VANE, Pharmacol. Rev. 30 293-331 (1979). S. MONCADA, E . A . HIGGS and J . R . VANE, Lancet i 18-21 (1977). J . A . SALMON, Prostaglandins 15 383-396 (1978). E.M. BROWN and S . L . SWARTZ, Prostaglandins 29 35-46 (1985). P . Y . K . WONG and W.Y. CHEUNG, Biochem. B i o p h y s . Res. Commun. 90 473-480 (1979). Y. SAGLIKES, S.G. MASSRY, K . , ISEKI, J . L . , NADLER AND V . M . CAMPESE, Am. J. Physiol. 248 F674-F681 (1985).