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Effect of bromocriptine on prostacyclin release and cyclic nucleotides on rat aortic and uterine tissues
PROSTAGLANDINS EFFECT OF BROMOCRIPTINEON PROSTACYCLINRELEASEAND CYCLIC NUCLEOTIDESON RAT AORTIC AND UTERINE TISSUES Ageel,
A.M.,
Department University,
El-Tahir,
K.E.H.
of Pharmacology, P.O. Box 2457,
and Abu-Jayyab,
A. R.
College of Pharmacy, Riyadh-11451, Saudi
King Saud arabia.
ABSTRACT The effect of bromocriptine mesylate on cyclic nucleotides and PGIz release by rat aortic and uterine tissues was investigated. Treatment of rats with bromocriptine (10 mg kg-l I.P. daily for 14 days) increased PGI, release by the thoracic aorta from 0.67 ? 0.02 to 1.4 f 0.03 ng/mg wet tissue (P(O.001; n = 6). This increase was antagonized by treatment with sulpiride (15 mg kg-l). Incubation of the arterial tissue with bromocriptine (50 ug ml-l) in vitro also stimulated PGI, release. Mepacrine (160 pg ml-l) significantly decreased both basal and stimulated PGI, release. Incubation of myomet rial tissue from pregnant rats with bromocriptine (50 pg ml-l) in vitro significantly decreased PGI, release from 1.25 + 0.07 to 0.60 + 0.08 ng/mg wet tissue (P
SEPTEMBER 1985 VOL. 30 NO. 3
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PROSTAGLANDINS METHODS Male Sprague-Dawleyrats (250 g) were divided into 4 groups (n = 6). The animals were injected I.P. as follows: group 1, bromocriptine mesylate 10 mg/kg; group 2, sulpiride 15 mg/kg; group 3, bromocriptine 10 mg/kg + sulpiride 15 mg/kg; group 4, vehicle. All drugs were dissolved in saline. A few drops of diluted HCl were used to enhance the solubility of sulpiride. The drugs were injected daily at 8:00 a.m. for 14 days. The last dose was injected 1 h before killing. The animals were anaesthetizedwith ether, 5 ml blood were removed from each animal by cardiac puncture in a lo-ml plastic syringe containing heparin (50 I.D/ml blood). The animals were then killed and the abdomen and thorax opened. The thoracic aorta was cleared from adhering tissues and fat removed and placed on ice-cold Ca-free Krebs' solution (pH 8). To study PGI, release, the tissues were blotted dry, 50 mg pieces were then cut into rings, placed in small plastic cuevettes and suspended in Ca-free Krebs' solution to give 25% w/v mixture. A small siliconized stainless steel stirrer was placed in the cuvette and the contents were incubated with stirring in an aggregometer chamber (H. Hupchurch 8 Co) at 37°C for 3 min. At the end of the incubation period, the cuevette was removed >nd placed on an ice bath. The PGI, content of the incubation medium was estimated against authentic PGI, using the platelet antiaggregatory assay using titrated rabbit platelets rich plasma as described previously (7). ADP was used as the aggregating agent (10 JIM final concentration). In some experiments aortic tissue was removed fro? control rats and incubated in presence of bromocriptine (5Oyg ml ), mepacrine 16Opg ml-l or a mixture of mepacrine and bromocriptine, PGI, release was then studied as above. Studies on myometrial PGI, release: Female Sprague-Dawley rats (250) were mated overnight and the following day the presence of a cervical plug was taken as evidence of fertilization. This day was denoted day 1 of pregnancy. Animals (n = 6) were killed on day 16 of pregnancy. The abdomen was opened and the two uterine horns were removed and placed on ice-cold Krebs' solution. After removal of the uterine contents, themyometrial areas which lay beneath the placentae were carefully dissected out and discarded as they don't produce PGI,. Decidual tissue was then separated from the myometrium (7) and discarded. 50 mg pieces of the myometrium were suspended in 0.2 ml Ca-free Krebs' solution in absence or presence of bromocriptinemesylate to give a final concentration of 50 pg ml-l. In other samples sulpiride (50 pg ml-l) or a mixture of bromocriptine and sulpiride was added. PGI, release was then studied as described above. Characterizationof the released antiaggregatorymaterial: Samples of the media in which the aortic and myometrid tissues were incubated were subjected to the following treatments:
370
SEPTEMBER
1985 VOL.30N0.3
PROSTAGLANDINS
a)
An aliquot of the incubation mediumthat showed antiaggregatory activity was heated at 37'C for 10 min and the antiaggregatory activity was retested.
b)
Other aliquots of the incubation mediumwere alkalinized to pH 10 with IN NaOH, placed on an ice bath for 60 min, then neutralized to pH 7.5 with IN HCl and the antiaggregatoryactivity was retested(7).
In some experiments control samples of aortic and myometrial tissues were incubated in presence of indomethacin 20 pg ml-l and the antiaggregatoryactivity was examined. Measurement of CAMP and cGMP in uterine and aortic tissues in vitro: Female rats were killed on Day 16 of pregnancy. Aortic and whole uterine tissues were removed as described above. Uterine tissue slices (30 mg each) were divided into 4 groups (n = 6). Each tissue was suspended in 1 ml Krebs-Kinger bicarbonate buffer (pH,7.4) and incubated for 60 min in a shaking water bath at 37'C with 2 changes of medium. Each tissue was then removed and placed in a small centrifuge tube containing 0.3 ml Krebs-Ringerbicarbonate buffer and 0.05 ml theophylline (2 mM final concentration). To each tube in group 1, bromocriptinemesylate (0.05 ml) to give a final concentration of 50 pg/ml was addedito group 2, a mixture of bromocriptine + sulpiride (0.05 ml) to give a final concentration of 50 pg each/ml was added; to group 3, sulpiride (0.05 ml) to give a final concentration of 50 pg/ml was added and to group 4, buffer (0.05 ml) was added. The same procedure was repeated using aortic slices 15 mg each, n = 6). All tubes were flushed for 1 min with 5% CO, in oxygen and sealed. The tubes were then incubated with shaking for 15 min at 37'C. Incubation was terminated by immersing the tubes in a boiling water bath for 10 min. The tubes were then centrifuged at 3000 rpm for 15 min. From each tube 2 x 0.05 ml supernatant aliquots were removed and assayed for CAMP content by the Amersham Kit procedure. Similarly 2 x 0.1 ml supernatant aliquots were assayed for cGMP content by the Amersham Kit procedure. The cyclic nucleotide content of the supernatants was calculated as p moles cyclic nucleotide/lOOmg wet tissue. Statistical significance of the results was calculated using Students' ‘t’ test (paired or unpaired as appropriate). Drugs used were: Bromocriptinemesylate (Sandoz), Sulpiride (Laboratoire Etudes et Development Chimiques, Arpajon, France), Prostacyclin (Wellcome Research Laboratories),ADP (BoehringerJmepacrine hydrochloride (Sigma), Indomethacin (MSD), other chemicals (BDH).
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PROSTAGLANDINS
RESULTS
Studies on aortic PGI, release.
A typical example of the effect of in vivo treatment with bromocriptine, sulpiride or their combinations on aortic PGI, release in vitro is shown in Figure 1. 15 ~1 aliquot of an incubation mxium of a control tissue contained an antiaggregatoryeffect equivalent to 2.5 ng PGI,; this corresponds to a synthesis of 0.66 ng/mg wet tissue over the 3 min incubation period. However, an equivalent volume of medium of a tissue taken from an animal treated with bromocriptine contained 5 ng PGI,,correspondingto a synthesis of 1.3 ng/mg, an increase of 100% over the control release. Equivalent aliquots of incubation media of tissues removed from animals treated with bromocriptine + sulpiride or sulpiride alone contained almost equal antiaggregatoryactivity as observed in the control tissue. The cumulative findings from a series of 6 experiments are illustrated in Figure 2. Chronic treatment with bromocriptine increased PG12 generation from 0.67 f 0.02 (Mean f SEM) (control) to 1.40 f 0.03 ng/mg wet tissue/3 min at 37OC. This increase is highly significant (P
372
SEPTEMBER 1985 VOL. 30 NO. 3
PROSTAGLANDINS
AI-ID
ADP
ADP
AOP ._... .
ADP
M
i I-
I
PGb
~2.Sng
!
C I!3
1 B
15Ul
1 PGh
5ng
E
I B+s
15Ul
1 2.
IIp
1’
PGh . .3C_^
Figure 1. The effect of bromocriptine,sulpiride + bromocriptine and sulpiride alone on rat aortic PGI, release in vitro. The change in light transmission caused by addition of ADP 10@1 (v) to rabbit titrated platelet rich plasma was recorded. Addition of ADP alone caused irreversibleplatelet aggregation. Addition of 15 ~'1aliquot of control incubation medium (C) decreased ADP-induced platelet aggregation due to the released PGI,. An equivalent aliquot from a tissue obtained from a rat treated with bromocriptine (B) (10 mg/kg I.P. for 14 days) contained twice as much antiaggregatoryactivity. Similar aliquots obtained from tissues of rats treated with bromocriptine + sulpiride (B + S) or sulpiride alone (S) (15 mg/kg I.P. for 14 days) contained antiaggregatoryactivity comparable to that from control tissue (C).
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1
BtS
Figure 2. Histogram showing the effect of in vivo treatment with bromocriptine,sulpiride+ bromocriptine andwide alone on rat aortic PGI, release in vitro. Control release of PGI, (C) was stimulated after pretreatment with bromocriptine (B) (10 mg/kg I.P. for 14 days). PGI, release by tissues obtained from rats pretreated with sulpiride+ bromocriptine (S + B) or sulpiride alone (S) (15 mg/ kg I.P. for 14 days) was similar to that from control tissues. Each column represents the mean from 6 experiments. Vertical lines represent s.e.m. Statistically significant increase is shown (*) (P(O.001).
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ADP t$O$M
v
B yw9
I2+5n9
sD’sAg
B 0'5/ug B O&g
M 10/u
(M+B) 10&l
I2 2.5ng
(t&S) IQAJ
M+S)W
S &fig
s o$_kj
Figure 3. The effect of bromocriptineupon PGI, output from myometrial tissue obtained from a 16-day pregnant rat. The increase in light transmission caused by addition of ADP 10 pM (v) to rabbit titrated platelet rich plasma (PRP) was recorded. Addition of 0.5 pg bromocriptine [B) plus 0.5 )lgsulpiride (S) to the PRP 1 min before ADP did not affect ADP-induced aggregation. As a safeguard 0.5 pg B and or 0.5 PgS were added 1 min before ADP as appropriate. 10 pl of the incubation medium in which the myometrium was incubated in presence of bromocriptine (M + B) contained less PGI, than the control myometrial incubate (M). 10 ~1 aliquots of media in which the myometrium was incubated in presence of sulpiride (M + S) or bromocriptine + sulpiride (M + B + S) contained nearly the same antiaggregatoryactivity as the control myometrium.
SEPTEMBER 1985 VOL. 30 NO. 3
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synthesis of 1 ng PGI, / mg, a decrease of 44% below the control release. Equivalent aliquots of incubate taken from media in which the myometrium was incubated in presence of sulpiride or a mixture of bromocriptine + sulpiride contained antiaggregatory activities similar to those observed in control samples. The cumulative findings from 6 experiments are shown in Table 1. As seen from the Table, incubation of the myometrial tissue with bromocriptine significantly decreased PGI, gemeration. Sulpiride did not affect basal PGIz release but completely abolished the inhibitory effect of bromocriptine. Table
1. Effect of bromocriptine, sulpiride and their combination on myometrial PGI, release, (ng/mg/3 min at 37’C, mean + SEM, n = 6).
Treatment
PGI, Production
Contro 1
1.25
Bromocriptine
0.6
? 0.08*
Sulpiride
1.4
f 0.1
Bromocriptine + Sulpiride
1.1
2 0.1
*
P < 0.05,
Effect of heat, antiaggregatory
(n = 6). alkaline activity:
Compared with
2 0.07
control.
pH and indomethacin
on the released
When aliquots of control incubation media were heated at 37’C for 10 min and their antiaggregatory activities were retested, no antiaggregatory activity was detected. When aliquots of control incubation media were alkalinized pH 10 and left to stand on an ice-bath for 1 hour, neutralized pH 7.5 and the antiaggregatory activity was retested, no loss ‘the antiaggregatory activity was observed. When aliquots of incubation media in which the tissues incubated with indomethacin were tested, no antiaggregatory vity was detected. Effect
on cyclic
a) Effect
376
to to of
were acti-
nucleotides:
on CAMP:
SEPTEMBER 1985 VOL. 30 NO. 3
PROSTAGLANDINS
Incubation of uterine tissue in presence of bromocriptine significantly increased uterine CAMP release from 40 _+ 2 (control to 64 + 3 p moles/100 mg wet tissue, a mean % increase of 60% (P
t f
Figure 4. Histogram showing the effect of bromocriptine (B), sulpiride (S) or their combination (B+S) on cAEIP release in vitro by ute--rine tissues obtained from 16-day pregnant rats. Basal CAMP release column) was significantly increased in presence of bromoCC; solid criptine 50 pg/ml (open column). The presence of sulpiride with bromocriptine (B + S) prevented the stimulant effect of bromocriptine. Sulpiride alone 50 pg/ml (S) did not affect basal CA!@ release. Vertical lines represent s.e.m. Significant difference is shown (*) (P(O.05; n = 6).
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PROSTAGLANDINS
with bromocriptine,sulpiride or their combination did not alter basal CAMP. The CAMP released by control tissues was 71 f 6 pmoles/ 100 mg wet tissue whereas that in presence of bromocriptine, sulpiride + bromocriptine and sulpiride alone was 60 + 5, 62 + 4 and 63 ?:4 p moles/100 mg respectively (n = 6). 2) Effect on cGMP: The effect of bromocriptine, sulpiride + bromocriptine and sulpiride alone on aortic and uterine cGMP release is shown in Table 2. As seen from the Table noneof the treatments affected basal cGMP release to any significant level. Table 2. Effect of bromocriptine, sulpiride + bromocriptine and sulpiride on aortic and uterine cGMP release (P moles/100 mg wet tissue, mean 2 SEM, n = 6).
Treatment
Aorta
Uterus
Vehicle
14.3 + 0.15
6.9 + 0.15 6.3 f 0.15
Bromocriptine
14.5 f 0.17
Sulpiride + Bromocriptine
13.8 f 0.14
Sulpiride
14.2 + 0.12
7.3 t 0.2
DISCUSSION: The results of these studies indicate that bromocriptine enhances release of an antiaggregatoryactivity from the rat thoracic aorta. This activity was characterized as PGI, because of its unstability at 37'C, stability in alkaline media, diminished release in presence of mepacrine, the phospholipaseA, inhibitor and indomethacin, the PG cyclooxygenase inhibitor. The increased PGI, release in presence of bromocriptine seems to be due to activation of phospholipase A, enzyme that stimulates the release of arachidonic acid, the precursor of PGI,. This suggestion is based on the finding that mepacrine significantly abolished the basal and the induced release. In this context it has been reported that bromocriptine increased plasma free fatty acids presumably by activating tissue lipases (9). The failure of bromocriptine to affect the release of both CAMP and cGMP by the aorta excludes the involvement of these nucleotides on the induced release of PGI,. The effectiveness of the dopamine antagonist sulpiride to prevent the induced release suggests
378
SEPTEMBER 1985 VOL. 30 NO. 3
PROSTAGLANDINS
that that
release was mediated via activation are not linked to adenylate cyclase
of dopamine D-2 receptors (10) *
The ability of bromocriptine to evoke PGI, release in arterial tissues may contribute to its induced relaxation in peripheral arteries (11). Thus, it may prove to be beneficial in some cases of blood flow insufficiencies. Incubation of myometrial tissue with bromocriptine significantly reduced the release of the antiaggregatory activity. This activity has been fully characterized as PGI, (7). The decrease in uteof uterine CAMP. High rine PGI, seems to be linked to the elevation levels of CAMP are known to decrease the availability of free Ca2+ ions resulting in decreased arachiodonic acid release (12). dopamine receptors are diviAccording to biochemical theories, ded into D-l receptors responsible for stimulating adenylate cyclase and D-2 receptors that may either decrease or have no effect on CAMP is supposed to be effective at D-2 receptors (14). (13) - Sulpiride Accordingly, the stimulant effect of bromocriptine on uterine CAMP was due to activation of D-l receptors. However, the successful antagonizm of the induced increase in CAMP and the decrease in myometrial PGI, by sulpiride questions the selectivity of this antagonist to D-2 receptors. Alternatively, the uterine dopamine receptors are of the D-2 type that are linked to adenylate cyclase. Bromocriptine-induced decrease in uterine PGI, may be beneficial to decrease postpartum haemorrhage when the compound is prescribed postpartum to inhibit lactation. Furthermore, the decrease in PGI, may ?lelp to alleviate some of the symptoms of premenstrual tension. El Tahir & Williams (15) and Phillips and Poyser (16) found a selective and a significant increase in PGI, in the implantation sites in rats. It was suggested that PGI, induced vasodilation may Thus, the inhibitory effect of bromoaid blastocyst establishment. criptine on myometrial PGI, may underlie the reported inhibitory effect of the compound on implantation in rats (17). the inhibitory effect of On a wider basis, metrial PGI, together with the luteolytic effect various mammals including humans (18, 19) point for bromocriptine in postcoital contraception.
bromocriptine on myoof bromocriptine in to a potential role
ACKNOWLEDGEMENT We would like to thank Sandoz for a gift of bromocriptine, Mr Abdalla Abd El-Razig for his technical assistance in cyclic nucleotides assay and Mr Barkatullah Javed for his assistance type-writing the manuscript.
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in
379
PROSTAGLANDINS REFERENCES: 1.
Johnson, A.M., D.M. Loew, & J.M. Vigouret, Stimulant properties of bromocriptine on central dopamine receptors in comparison to apomorphine,(+)-amphetamine and L-dopa, Br.J.Pharmac.s:59,1976.
2.
Brodde, O.E., J. Freistuehler 4 F.J. Meyer Sterespecific antagonizm by D-butacalmol of dopamine-inducedrelaxation of the isolated rabbit mesentric artery. J. Cardiovasc. Pharmacol. -3: 828, 1981.
3.
Omini, C. S. Moncada, J.R. Vane. The effect of Prostacyclin on tissues which detect prostaglandins. Prostaglandins,-14: 625, 1977.
4.
Clarke, B.J., G. Scholtysik FrE. FlGckiger, Cardiovascular actions of bromocriptine. Acta Endocr. (Suppl. 216) -88: 75, 1978.
5.
Armstrong, J.M., D. Chapple, G.J. Dusting, R. Hughes, S. Moncada & J.R. Vane. Cardiovascularactions of prostacyclin in chloralose anaesthetizeddogs. Br. J. Pharmac. -61: 136P, 1977.
6.
El Tahir, K.E.H. 6 K.I. Williams Oxytocic drugs stimulate prostacyclin release from the rat pregnant myometrium. Br. J. Pharmat. 70: 86 P, 1980.
7.
El Tahir, K.E.H. & K.I. Williams. Factors affecting prostacyclin formation by the rat pregnant myometrium. Br. J. Pharmac. -71: 641, 1980.
8.
Ali, M.B., K.E. El Tahir & K.I. Williams. Influence of pregnancy and ovarian steroids on myometrial and vascular prostacyclin product_ion. In: Prostacyclin in pregnancy (P.J. Lewis, S.Moncada E J.O. Grady; eds). Raven Press, New York, 1983, p 131. Tolis, G., E.J. Pinter G H.G. Friesen. Acute effects of 2-bromoP(-erogcriptine (CB-154) on anterior pituitary hormones and free fatty acids in man. Int. J. Clin. Pharmacol. Biopharm. -12: 281, 1975.
9.
10. Kebabian, J.W. & D.B. Calne, Multiple receptors for dopamine. Nature 277: 93, 1979. 11. Oudart, N., P. Aubineau, R. Sercombe, R. Boulu 4 J. Seylaz. Dopaminergic relaxation in cerebral and peripheral arteries in vitro. Dev. Neurosci (Amesterdam)-14: 153, 1982. 12. Gerrard, J.M., J.D. Peller, T.P. Krick, 6 J.G. White. Cyclic AMP and platelet prostaglandin synthesis. Prostaglandins-14: 39, 1977.
380
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13.
C.W. Grewe & J.W. Kebabian. Stimulation of the D-2 Cote, T.E., Dopamine receptor in the intermediate lobe of the rat pituitary glands decreases the responsiveness of the betaadrenoceptor: biochemical mechanism. Endocrinology -108: 420, 1981.
14.
Agonist Creese, I , D.R. Sibley 6 S.E. Leff. dopamine receptors: focus on radioligand-binding Proc. -43: 2779, 1984.
15.
El Tahir, K.E.H. & K.I. Williams. thesis - a new factor in pregnancy (London) 296: 86P, 1979.
16.
Phillips, C.R. 6 N.L. Poyser. Prostaglandins in the rat. Adv. Prostaglandins Thromboxane
17.
T. Bauknecht i!, J.W. Siebers. The effect of Mueller, U., criptine during early pregnancy in the rat: inhibition of plantation. Acta Endocrinol. (Copenhagen) -94: 268, 1980.
18.
Daniel, C.V. E Goldziehr, J.W. The Luteolytic and abortifacient potential of an oestrogen-bromoergocriptine regimen in the baboon. Fertil. Steril. -37: 258, 1982.
19.
Blackwell, R.E., L.R. Boots 6 H.D., Jr. delestrogen and parlodel as a luteolytic Fertil. Steril. -37: 213, 1982. Editor:
H. R.
Behrman
Received:
SEPTEMBER 1985 VOL. 30 NO. 3
interactions studies.
with Fed.
Myometrial prostacyclin in the rat? J. Physiol.
syn-
in implantation Res. - 8: 139, 1980. bromoim-
Potter. Evaluation agent in humans.
3-11-85
Accented
of
: 6-ll-
85
381
A.M.,
Department University,
El-Tahir,
K.E.H.
of Pharmacology, P.O. Box 2457,
and Abu-Jayyab,
A. R.
College of Pharmacy, Riyadh-11451, Saudi
King Saud arabia.
ABSTRACT The effect of bromocriptine mesylate on cyclic nucleotides and PGIz release by rat aortic and uterine tissues was investigated. Treatment of rats with bromocriptine (10 mg kg-l I.P. daily for 14 days) increased PGI, release by the thoracic aorta from 0.67 ? 0.02 to 1.4 f 0.03 ng/mg wet tissue (P(O.001; n = 6). This increase was antagonized by treatment with sulpiride (15 mg kg-l). Incubation of the arterial tissue with bromocriptine (50 ug ml-l) in vitro also stimulated PGI, release. Mepacrine (160 pg ml-l) significantly decreased both basal and stimulated PGI, release. Incubation of myomet rial tissue from pregnant rats with bromocriptine (50 pg ml-l) in vitro significantly decreased PGI, release from 1.25 + 0.07 to 0.60 + 0.08 ng/mg wet tissue (P
SEPTEMBER 1985 VOL. 30 NO. 3
369
PROSTAGLANDINS METHODS Male Sprague-Dawleyrats (250 g) were divided into 4 groups (n = 6). The animals were injected I.P. as follows: group 1, bromocriptine mesylate 10 mg/kg; group 2, sulpiride 15 mg/kg; group 3, bromocriptine 10 mg/kg + sulpiride 15 mg/kg; group 4, vehicle. All drugs were dissolved in saline. A few drops of diluted HCl were used to enhance the solubility of sulpiride. The drugs were injected daily at 8:00 a.m. for 14 days. The last dose was injected 1 h before killing. The animals were anaesthetizedwith ether, 5 ml blood were removed from each animal by cardiac puncture in a lo-ml plastic syringe containing heparin (50 I.D/ml blood). The animals were then killed and the abdomen and thorax opened. The thoracic aorta was cleared from adhering tissues and fat removed and placed on ice-cold Ca-free Krebs' solution (pH 8). To study PGI, release, the tissues were blotted dry, 50 mg pieces were then cut into rings, placed in small plastic cuevettes and suspended in Ca-free Krebs' solution to give 25% w/v mixture. A small siliconized stainless steel stirrer was placed in the cuvette and the contents were incubated with stirring in an aggregometer chamber (H. Hupchurch 8 Co) at 37°C for 3 min. At the end of the incubation period, the cuevette was removed >nd placed on an ice bath. The PGI, content of the incubation medium was estimated against authentic PGI, using the platelet antiaggregatory assay using titrated rabbit platelets rich plasma as described previously (7). ADP was used as the aggregating agent (10 JIM final concentration). In some experiments aortic tissue was removed fro? control rats and incubated in presence of bromocriptine (5Oyg ml ), mepacrine 16Opg ml-l or a mixture of mepacrine and bromocriptine, PGI, release was then studied as above. Studies on myometrial PGI, release: Female Sprague-Dawley rats (250) were mated overnight and the following day the presence of a cervical plug was taken as evidence of fertilization. This day was denoted day 1 of pregnancy. Animals (n = 6) were killed on day 16 of pregnancy. The abdomen was opened and the two uterine horns were removed and placed on ice-cold Krebs' solution. After removal of the uterine contents, themyometrial areas which lay beneath the placentae were carefully dissected out and discarded as they don't produce PGI,. Decidual tissue was then separated from the myometrium (7) and discarded. 50 mg pieces of the myometrium were suspended in 0.2 ml Ca-free Krebs' solution in absence or presence of bromocriptinemesylate to give a final concentration of 50 pg ml-l. In other samples sulpiride (50 pg ml-l) or a mixture of bromocriptine and sulpiride was added. PGI, release was then studied as described above. Characterizationof the released antiaggregatorymaterial: Samples of the media in which the aortic and myometrid tissues were incubated were subjected to the following treatments:
370
SEPTEMBER
1985 VOL.30N0.3
PROSTAGLANDINS
a)
An aliquot of the incubation mediumthat showed antiaggregatory activity was heated at 37'C for 10 min and the antiaggregatory activity was retested.
b)
Other aliquots of the incubation mediumwere alkalinized to pH 10 with IN NaOH, placed on an ice bath for 60 min, then neutralized to pH 7.5 with IN HCl and the antiaggregatoryactivity was retested(7).
In some experiments control samples of aortic and myometrial tissues were incubated in presence of indomethacin 20 pg ml-l and the antiaggregatoryactivity was examined. Measurement of CAMP and cGMP in uterine and aortic tissues in vitro: Female rats were killed on Day 16 of pregnancy. Aortic and whole uterine tissues were removed as described above. Uterine tissue slices (30 mg each) were divided into 4 groups (n = 6). Each tissue was suspended in 1 ml Krebs-Kinger bicarbonate buffer (pH,7.4) and incubated for 60 min in a shaking water bath at 37'C with 2 changes of medium. Each tissue was then removed and placed in a small centrifuge tube containing 0.3 ml Krebs-Ringerbicarbonate buffer and 0.05 ml theophylline (2 mM final concentration). To each tube in group 1, bromocriptinemesylate (0.05 ml) to give a final concentration of 50 pg/ml was addedito group 2, a mixture of bromocriptine + sulpiride (0.05 ml) to give a final concentration of 50 pg each/ml was added; to group 3, sulpiride (0.05 ml) to give a final concentration of 50 pg/ml was added and to group 4, buffer (0.05 ml) was added. The same procedure was repeated using aortic slices 15 mg each, n = 6). All tubes were flushed for 1 min with 5% CO, in oxygen and sealed. The tubes were then incubated with shaking for 15 min at 37'C. Incubation was terminated by immersing the tubes in a boiling water bath for 10 min. The tubes were then centrifuged at 3000 rpm for 15 min. From each tube 2 x 0.05 ml supernatant aliquots were removed and assayed for CAMP content by the Amersham Kit procedure. Similarly 2 x 0.1 ml supernatant aliquots were assayed for cGMP content by the Amersham Kit procedure. The cyclic nucleotide content of the supernatants was calculated as p moles cyclic nucleotide/lOOmg wet tissue. Statistical significance of the results was calculated using Students' ‘t’ test (paired or unpaired as appropriate). Drugs used were: Bromocriptinemesylate (Sandoz), Sulpiride (Laboratoire Etudes et Development Chimiques, Arpajon, France), Prostacyclin (Wellcome Research Laboratories),ADP (BoehringerJmepacrine hydrochloride (Sigma), Indomethacin (MSD), other chemicals (BDH).
SEPTEMBER 1985 VOL. 30 NO. 3
371
PROSTAGLANDINS
RESULTS
Studies on aortic PGI, release.
A typical example of the effect of in vivo treatment with bromocriptine, sulpiride or their combinations on aortic PGI, release in vitro is shown in Figure 1. 15 ~1 aliquot of an incubation mxium of a control tissue contained an antiaggregatoryeffect equivalent to 2.5 ng PGI,; this corresponds to a synthesis of 0.66 ng/mg wet tissue over the 3 min incubation period. However, an equivalent volume of medium of a tissue taken from an animal treated with bromocriptine contained 5 ng PGI,,correspondingto a synthesis of 1.3 ng/mg, an increase of 100% over the control release. Equivalent aliquots of incubation media of tissues removed from animals treated with bromocriptine + sulpiride or sulpiride alone contained almost equal antiaggregatoryactivity as observed in the control tissue. The cumulative findings from a series of 6 experiments are illustrated in Figure 2. Chronic treatment with bromocriptine increased PG12 generation from 0.67 f 0.02 (Mean f SEM) (control) to 1.40 f 0.03 ng/mg wet tissue/3 min at 37OC. This increase is highly significant (P
372
SEPTEMBER 1985 VOL. 30 NO. 3
PROSTAGLANDINS
AI-ID
ADP
ADP
AOP ._... .
ADP
M
i I-
I
PGb
~2.Sng
!
C I!3
1 B
15Ul
1 PGh
5ng
E
I B+s
15Ul
1 2.
IIp
1’
PGh . .3C_^
Figure 1. The effect of bromocriptine,sulpiride + bromocriptine and sulpiride alone on rat aortic PGI, release in vitro. The change in light transmission caused by addition of ADP 10@1 (v) to rabbit titrated platelet rich plasma was recorded. Addition of ADP alone caused irreversibleplatelet aggregation. Addition of 15 ~'1aliquot of control incubation medium (C) decreased ADP-induced platelet aggregation due to the released PGI,. An equivalent aliquot from a tissue obtained from a rat treated with bromocriptine (B) (10 mg/kg I.P. for 14 days) contained twice as much antiaggregatoryactivity. Similar aliquots obtained from tissues of rats treated with bromocriptine + sulpiride (B + S) or sulpiride alone (S) (15 mg/kg I.P. for 14 days) contained antiaggregatoryactivity comparable to that from control tissue (C).
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1
BtS
Figure 2. Histogram showing the effect of in vivo treatment with bromocriptine,sulpiride+ bromocriptine andwide alone on rat aortic PGI, release in vitro. Control release of PGI, (C) was stimulated after pretreatment with bromocriptine (B) (10 mg/kg I.P. for 14 days). PGI, release by tissues obtained from rats pretreated with sulpiride+ bromocriptine (S + B) or sulpiride alone (S) (15 mg/ kg I.P. for 14 days) was similar to that from control tissues. Each column represents the mean from 6 experiments. Vertical lines represent s.e.m. Statistically significant increase is shown (*) (P(O.001).
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ADP t$O$M
v
B yw9
I2+5n9
sD’sAg
B 0'5/ug B O&g
M 10/u
(M+B) 10&l
I2 2.5ng
(t&S) IQAJ
M+S)W
S &fig
s o$_kj
Figure 3. The effect of bromocriptineupon PGI, output from myometrial tissue obtained from a 16-day pregnant rat. The increase in light transmission caused by addition of ADP 10 pM (v) to rabbit titrated platelet rich plasma (PRP) was recorded. Addition of 0.5 pg bromocriptine [B) plus 0.5 )lgsulpiride (S) to the PRP 1 min before ADP did not affect ADP-induced aggregation. As a safeguard 0.5 pg B and or 0.5 PgS were added 1 min before ADP as appropriate. 10 pl of the incubation medium in which the myometrium was incubated in presence of bromocriptine (M + B) contained less PGI, than the control myometrial incubate (M). 10 ~1 aliquots of media in which the myometrium was incubated in presence of sulpiride (M + S) or bromocriptine + sulpiride (M + B + S) contained nearly the same antiaggregatoryactivity as the control myometrium.
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synthesis of 1 ng PGI, / mg, a decrease of 44% below the control release. Equivalent aliquots of incubate taken from media in which the myometrium was incubated in presence of sulpiride or a mixture of bromocriptine + sulpiride contained antiaggregatory activities similar to those observed in control samples. The cumulative findings from 6 experiments are shown in Table 1. As seen from the Table, incubation of the myometrial tissue with bromocriptine significantly decreased PGI, gemeration. Sulpiride did not affect basal PGIz release but completely abolished the inhibitory effect of bromocriptine. Table
1. Effect of bromocriptine, sulpiride and their combination on myometrial PGI, release, (ng/mg/3 min at 37’C, mean + SEM, n = 6).
Treatment
PGI, Production
Contro 1
1.25
Bromocriptine
0.6
? 0.08*
Sulpiride
1.4
f 0.1
Bromocriptine + Sulpiride
1.1
2 0.1
*
P < 0.05,
Effect of heat, antiaggregatory
(n = 6). alkaline activity:
Compared with
2 0.07
control.
pH and indomethacin
on the released
When aliquots of control incubation media were heated at 37’C for 10 min and their antiaggregatory activities were retested, no antiaggregatory activity was detected. When aliquots of control incubation media were alkalinized pH 10 and left to stand on an ice-bath for 1 hour, neutralized pH 7.5 and the antiaggregatory activity was retested, no loss ‘the antiaggregatory activity was observed. When aliquots of incubation media in which the tissues incubated with indomethacin were tested, no antiaggregatory vity was detected. Effect
on cyclic
a) Effect
376
to to of
were acti-
nucleotides:
on CAMP:
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Incubation of uterine tissue in presence of bromocriptine significantly increased uterine CAMP release from 40 _+ 2 (control to 64 + 3 p moles/100 mg wet tissue, a mean % increase of 60% (P
t f
Figure 4. Histogram showing the effect of bromocriptine (B), sulpiride (S) or their combination (B+S) on cAEIP release in vitro by ute--rine tissues obtained from 16-day pregnant rats. Basal CAMP release column) was significantly increased in presence of bromoCC; solid criptine 50 pg/ml (open column). The presence of sulpiride with bromocriptine (B + S) prevented the stimulant effect of bromocriptine. Sulpiride alone 50 pg/ml (S) did not affect basal CA!@ release. Vertical lines represent s.e.m. Significant difference is shown (*) (P(O.05; n = 6).
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with bromocriptine,sulpiride or their combination did not alter basal CAMP. The CAMP released by control tissues was 71 f 6 pmoles/ 100 mg wet tissue whereas that in presence of bromocriptine, sulpiride + bromocriptine and sulpiride alone was 60 + 5, 62 + 4 and 63 ?:4 p moles/100 mg respectively (n = 6). 2) Effect on cGMP: The effect of bromocriptine, sulpiride + bromocriptine and sulpiride alone on aortic and uterine cGMP release is shown in Table 2. As seen from the Table noneof the treatments affected basal cGMP release to any significant level. Table 2. Effect of bromocriptine, sulpiride + bromocriptine and sulpiride on aortic and uterine cGMP release (P moles/100 mg wet tissue, mean 2 SEM, n = 6).
Treatment
Aorta
Uterus
Vehicle
14.3 + 0.15
6.9 + 0.15 6.3 f 0.15
Bromocriptine
14.5 f 0.17
Sulpiride + Bromocriptine
13.8 f 0.14
Sulpiride
14.2 + 0.12
7.3 t 0.2
DISCUSSION: The results of these studies indicate that bromocriptine enhances release of an antiaggregatoryactivity from the rat thoracic aorta. This activity was characterized as PGI, because of its unstability at 37'C, stability in alkaline media, diminished release in presence of mepacrine, the phospholipaseA, inhibitor and indomethacin, the PG cyclooxygenase inhibitor. The increased PGI, release in presence of bromocriptine seems to be due to activation of phospholipase A, enzyme that stimulates the release of arachidonic acid, the precursor of PGI,. This suggestion is based on the finding that mepacrine significantly abolished the basal and the induced release. In this context it has been reported that bromocriptine increased plasma free fatty acids presumably by activating tissue lipases (9). The failure of bromocriptine to affect the release of both CAMP and cGMP by the aorta excludes the involvement of these nucleotides on the induced release of PGI,. The effectiveness of the dopamine antagonist sulpiride to prevent the induced release suggests
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PROSTAGLANDINS
that that
release was mediated via activation are not linked to adenylate cyclase
of dopamine D-2 receptors (10) *
The ability of bromocriptine to evoke PGI, release in arterial tissues may contribute to its induced relaxation in peripheral arteries (11). Thus, it may prove to be beneficial in some cases of blood flow insufficiencies. Incubation of myometrial tissue with bromocriptine significantly reduced the release of the antiaggregatory activity. This activity has been fully characterized as PGI, (7). The decrease in uteof uterine CAMP. High rine PGI, seems to be linked to the elevation levels of CAMP are known to decrease the availability of free Ca2+ ions resulting in decreased arachiodonic acid release (12). dopamine receptors are diviAccording to biochemical theories, ded into D-l receptors responsible for stimulating adenylate cyclase and D-2 receptors that may either decrease or have no effect on CAMP is supposed to be effective at D-2 receptors (14). (13) - Sulpiride Accordingly, the stimulant effect of bromocriptine on uterine CAMP was due to activation of D-l receptors. However, the successful antagonizm of the induced increase in CAMP and the decrease in myometrial PGI, by sulpiride questions the selectivity of this antagonist to D-2 receptors. Alternatively, the uterine dopamine receptors are of the D-2 type that are linked to adenylate cyclase. Bromocriptine-induced decrease in uterine PGI, may be beneficial to decrease postpartum haemorrhage when the compound is prescribed postpartum to inhibit lactation. Furthermore, the decrease in PGI, may ?lelp to alleviate some of the symptoms of premenstrual tension. El Tahir & Williams (15) and Phillips and Poyser (16) found a selective and a significant increase in PGI, in the implantation sites in rats. It was suggested that PGI, induced vasodilation may Thus, the inhibitory effect of bromoaid blastocyst establishment. criptine on myometrial PGI, may underlie the reported inhibitory effect of the compound on implantation in rats (17). the inhibitory effect of On a wider basis, metrial PGI, together with the luteolytic effect various mammals including humans (18, 19) point for bromocriptine in postcoital contraception.
bromocriptine on myoof bromocriptine in to a potential role
ACKNOWLEDGEMENT We would like to thank Sandoz for a gift of bromocriptine, Mr Abdalla Abd El-Razig for his technical assistance in cyclic nucleotides assay and Mr Barkatullah Javed for his assistance type-writing the manuscript.
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in
379
PROSTAGLANDINS REFERENCES: 1.
Johnson, A.M., D.M. Loew, & J.M. Vigouret, Stimulant properties of bromocriptine on central dopamine receptors in comparison to apomorphine,(+)-amphetamine and L-dopa, Br.J.Pharmac.s:59,1976.
2.
Brodde, O.E., J. Freistuehler 4 F.J. Meyer Sterespecific antagonizm by D-butacalmol of dopamine-inducedrelaxation of the isolated rabbit mesentric artery. J. Cardiovasc. Pharmacol. -3: 828, 1981.
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Omini, C. S. Moncada, J.R. Vane. The effect of Prostacyclin on tissues which detect prostaglandins. Prostaglandins,-14: 625, 1977.
4.
Clarke, B.J., G. Scholtysik FrE. FlGckiger, Cardiovascular actions of bromocriptine. Acta Endocr. (Suppl. 216) -88: 75, 1978.
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Armstrong, J.M., D. Chapple, G.J. Dusting, R. Hughes, S. Moncada & J.R. Vane. Cardiovascularactions of prostacyclin in chloralose anaesthetizeddogs. Br. J. Pharmac. -61: 136P, 1977.
6.
El Tahir, K.E.H. 6 K.I. Williams Oxytocic drugs stimulate prostacyclin release from the rat pregnant myometrium. Br. J. Pharmat. 70: 86 P, 1980.
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El Tahir, K.E.H. & K.I. Williams. Factors affecting prostacyclin formation by the rat pregnant myometrium. Br. J. Pharmac. -71: 641, 1980.
8.
Ali, M.B., K.E. El Tahir & K.I. Williams. Influence of pregnancy and ovarian steroids on myometrial and vascular prostacyclin product_ion. In: Prostacyclin in pregnancy (P.J. Lewis, S.Moncada E J.O. Grady; eds). Raven Press, New York, 1983, p 131. Tolis, G., E.J. Pinter G H.G. Friesen. Acute effects of 2-bromoP(-erogcriptine (CB-154) on anterior pituitary hormones and free fatty acids in man. Int. J. Clin. Pharmacol. Biopharm. -12: 281, 1975.
9.
10. Kebabian, J.W. & D.B. Calne, Multiple receptors for dopamine. Nature 277: 93, 1979. 11. Oudart, N., P. Aubineau, R. Sercombe, R. Boulu 4 J. Seylaz. Dopaminergic relaxation in cerebral and peripheral arteries in vitro. Dev. Neurosci (Amesterdam)-14: 153, 1982. 12. Gerrard, J.M., J.D. Peller, T.P. Krick, 6 J.G. White. Cyclic AMP and platelet prostaglandin synthesis. Prostaglandins-14: 39, 1977.
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C.W. Grewe & J.W. Kebabian. Stimulation of the D-2 Cote, T.E., Dopamine receptor in the intermediate lobe of the rat pituitary glands decreases the responsiveness of the betaadrenoceptor: biochemical mechanism. Endocrinology -108: 420, 1981.
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Agonist Creese, I , D.R. Sibley 6 S.E. Leff. dopamine receptors: focus on radioligand-binding Proc. -43: 2779, 1984.
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El Tahir, K.E.H. & K.I. Williams. thesis - a new factor in pregnancy (London) 296: 86P, 1979.
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Phillips, C.R. 6 N.L. Poyser. Prostaglandins in the rat. Adv. Prostaglandins Thromboxane
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T. Bauknecht i!, J.W. Siebers. The effect of Mueller, U., criptine during early pregnancy in the rat: inhibition of plantation. Acta Endocrinol. (Copenhagen) -94: 268, 1980.
18.
Daniel, C.V. E Goldziehr, J.W. The Luteolytic and abortifacient potential of an oestrogen-bromoergocriptine regimen in the baboon. Fertil. Steril. -37: 258, 1982.
19.
Blackwell, R.E., L.R. Boots 6 H.D., Jr. delestrogen and parlodel as a luteolytic Fertil. Steril. -37: 213, 1982. Editor:
H. R.
Behrman
Received:
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interactions studies.
with Fed.
Myometrial prostacyclin in the rat? J. Physiol.
syn-
in implantation Res. - 8: 139, 1980. bromoim-
Potter. Evaluation agent in humans.
3-11-85
Accented
of
: 6-ll-
85
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