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Nitric oxide mediates plateletactivating factor stimulatory action on uterine prostaglandin production
Prostaglandins, Leukotrienssand Essential Fatty Acids(1998) 58(1), 55-59 © HarcourtBrace & Co Ltd 1998
Nitric oxide mediates platelet. activating factor stimulatory action on uterine prostaglandin production M. A. Chaud, A. M. Franchi, M. Ber6n de Astrada, M. F. Gimeno Centro de Estudios Farmacol6gicos y Botdnicos, Consejo Nacional de Investigaciones Cientificas y T~cnicas Serrano 669, 1414, Buenos Aires, Argentina
Accumulated evidence suggests that platelet-activating factor (PAF) may have a role in implantation by stimulating prostaglandin (PG) production. Since we had demonstrated that nitric oxide (NO) can increase uterine PGs, the aim of this study was to explore whether or not NO could mediate rat uterus responses to PAF on day 5 of gestation, when implantation takes place. Uterine motility was enhanced by PAF as compared to controls. This action was abolished by either the arginine analogue, N-monomethyl L-arginine (L-NMMA) or the cyclooxygenase inhibitor, indomethacin. On the other hand, NOS activity was detected in uterine strips and could be stimulated by PAF. The cyclooxygenase product PGE2 was also significantly stimulated by PAF. Inhibition of endogenous NO formation abolished the PAF effect on PG synthesis. Our results suggest that NO is an important intermediate in the interaction between PAF and PGs. Summary
INTRODUCTION
Increasing embryonic platelet-activating factor (PAF) production in the developing embryo prior to implantation might act as one of the initial signals that precedes the increase in vascular permeability and vasodflatation in the endometrium) -6 Local vasodilatation within the uterus is essential for the initiation of successful implantation, z Prostaglandins (PGs) have been proposed as the mediators of vascular permeability but also as required components of the progression of decidualization. 8,9 PAF binding sites are predominantly located in luminal epithelium 1° and it has been shown that PAF causes an increase in PG synthesis in the glandular fraction of the endometrium. 11,12 Nitric oxide (NO) is a gas molecule known to play an important role in many biological systems. Potential roles for NO on uterine function include vasodilatation and modulation of myometrial contractility, m4 We have demonstrated nerve fibers synthesizing NO in the uterus 15and showed that uterine NO has complex opposReceived 8 October 1996 Accepted 12 May 1997 Correspondence to: M. F. Gimeno, Fax. 00 54 01 856 2751
ing actions, relaxing uterine muscle by cGMP and contracting it via stimulation of the cyclooxygenase (COX) pathway, producing various prostanoids. 16 The present study was undertaken to examine a possible role of NO as a mediator of PAF actions in the rat uterus at day 5 of gestation. MATERIALS AND M E T H O D S Animals
Female rats of the Wistar strain (200-230 g body weight) were used. They were housed in group cages under controlled conditions of light (lights on 05:00-17:00 h) and temperature (23-25°C). Rat chow and water were freely available. Mating was confirmed by the presence of sperm in vaginal smears the next moming and this was considered to be the first day of gestation. Uterine tissue was obtained from rats at day 5 of gestation. Animals were killed by a blow on the neck and their uterine horns were removed. Fetuses, placenta and fetal membranes were separated and discarded. Metabolism of
[14C] arachidonic acid
Once the uterine horns were obtained, each horn was opened, trimmed of visible fat, and placed in a Petri dish 55
56
Chaud et al
containing a modified Krebs-Ringer bicarbonate (KRB) solution with glucose (11 raM). 1 The metabolism of exogenous arachidonic acid by rat uterine tissue was determined by incubating the tissue for 60 rain in KRB medium containing 0.25 ~tCi of [1'C] arachidonic acid (55 mCi/mol) in an atmosphere of 95% O2/CO2 with constant shaking at 60 cycles per rain at 37"C. For each determination, 200 nag of uterine tissue was used. The uterine strips were randomly treated with PAF (0.1 laM) or NG-monomethyl-L-arginine (NMMA) (300 glVl). These reagents were added to the incubation medium alone or in combination as described in the Results. The controls were incubated in medium alone. At the end of the incubation period the incubation medium was acidified to pH 3.0 with 1.0 M HCI in 1 vol of ethyl acetate and extracted twice for PGs. Pooled ethyl acetate extracts were dried under nitrogen. The residues were suspended in chloroform/methanol and applied to silica gel TIC plates. The plates were developed in a solvent system of benzene/ dioxane/glacial acetic acid (60:30:3; vol/vol). The position of the authentic eicosanoids was visualized by spraying the dried plates with 10% phosphomolybdic acid in ethanol followed by heating at 110°C for 10 min. Average Rf values were 0.35 for PGF2~, 0.47 for PGE2 and 0.8 for arachidonic acid. Radioactivity from TLC zones for arachidonic acid and for different prostanoids was measured by liquid scintillation counting. The area of each of the radioactive peaks corresponding to authentic prostanoids was calculated and expressed as a percentage of the total radioactivity of the plates.
NOS enzyme activity was quantified by a modified method of Bredt and Snyder, 1 which measures the conversion of [14C]-arginine into [14C)-citrulline. Slices were incubated at 37°C in a buffer that contained 20 nM HEPES, 10 ~VI [14C]-arginine (0.3 ~tCi) and 0.5 mn NADPH. After 15 min of incubation, samples were homogenized using a Tissuemizer. The samples were centrifuged for 10 minutes at 10 000 rpm and then they were applied to a 1 ml DOWEX AG5OW-X* column (Na+-form) and [~4C]citruUine was eluted in 3 ml of water. The radioactivity was measured by liquid scintillation counting. Enzyme activity is reported in cpm citrulline/1 O0 mg wet weight.
Motility studies
Statistics
Uterine horns were obtained as described above. The tissue was removed and each horn was divided by a transverse cut into two equal-length segments. The segments were placed in Petri dishes containing KRB at room temperature and constantly gassed with 95% OJCO2. Each segment was immediately opened by a cut along the mesosalpinx insertion; one end was attached to a glass holder and immersed in a tissue chamber filled with 20 ml of KRB (pH 7.4 37°C) and continuously gassed. The other end of the tissue was attached to a strain gauge coupled to an amplifier driving a direct writing oscillograph. After a resting tension of 1 g was applied to each strip by means of a micrometric device, isometric developed tension (IDT) and frequency of contractions (FC) were measured. IDT values (expressed in nag) were obtained by measuring the mean amplitude of all the contractions recorded over a 10-min period. FC values were obtained as the mean number of the contractile cycles observed during the same period. All uterine strips were preincubated in the chambers without tension, in the presence of NMMA (300 pM), indomethacin (1 pM) or
All values presented in this study represent means + SEM. Comparisons between groups were performed employing the ANOVA test. Differences between means were considered significant when Pwas 0.05 or less.
in an equal volume of medium alone for 30 min (control). After uteri preincubation a single dose of PAF (0.1 ~VI) was added. Uterine contractions in the first 10 min were taken as control. IDT and FC variations during the incubation period were expressed as a percentage change from the control. NOS enzyme assay
Drugs and chemicals
[1-14C]-arachidonic acid was provided by New England Nuclear (specific activity 56 ~tCi/mmol); L-[U14C]-arginine monohydrochloride was purchased from Amersham (specific activity 317 mCi/mmol); DOWEX AG5) W-X8 column (Na form) was provided by Bio Rad. All other reagents were obtained from Sigma Chemical Company.
RESULTS Effect of L-NMMA or indomethacin on PAF-induced motility
Figures 1-3 show the variations in uterine spontaneous motility during 40 min incubation. Data were expressed as percentage of change from the initial IDT (see Material and methods). FC values were also evaluated. Since the results obtained in FC followed the same patterns as IDT these data were not shown. As can be seen in Figure 1, the IDT of control uterine strips had an appreciable decay over time (60% after 40 min incubation). Uterine strips preincubated with the NOS inhibitor, L-NMMA showed considerably less change over 40 rain (30% decay after 40 roan). Tissues
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 55-59
© Harcourt Brace & Co Ltd 1998
NO mediates PAF stimu/atory action on uterine PG production
o PAF
= CONTROL Q NIIIt1~
0'
c-
"
a
0
" 1141)0
-20.
57
-G =
rtl111A+Pgr
, ]lll)0+PIg
-ZO.
c
-40.
~'
-40"
o
-60.
o
-6D~
#__
-80.
'-'
-I00
0
10
~0 ,~0 l]14E(m]n)
-80. -1OD 0
40
Fig. 1 Effect of 30 min preincubation with L-NMMA (300 I~M) or INDO (1 I~M) on the time course of IDT in uterine strips of pregnant rats. Points represent mean ± SEM of six uterine strips. **P < 0.01, *P < 0.05.
• CONIR0t o PAF
10
~'0 30 lillE(mini
40
Fig. 3 Effect of 30 min preincubation with L-NMMA (300 IIM) or INDO (1 I~M) on the time course of PAF (10 ~M) on the time course of IDT in uterine strips of pregnant rats. Points represent mean ± SEM of six uterine strips. **P < 0.01.
Table Effect of 30 min preincubation with L-NMMA (300 IIM) or INDO (100 llM) on initial IDT induced by PAF (10 llM) Initial IDT
-20.
Control INDO NMMA
~J cos cf=J
-40.
42 + 5 (6) 37 ± 4 (6) 28 ± 4 (6)
NOS activity was assessed in uterine strips by measuring the conversion of L-arginine to L-citrulline. As shown in Figure 4, uterine NOS activity was detected in 5 day pregnant rats and could be enhanced by PAF.
I--
-80]. 0
PAF INDO + PAF NMMA + PAF
Effect of PAF on uterine N O S activity
-60.
-100
30 + 4 (6) 35 + 3 (6) 35 ± 6 (6)
Ib
i0
4b
Effect of L - N M M A on the production of PGs from labelled A A in response to PAF
l]ME(miB) Fig. 2 Effect of PAF (10 ~M) on the time course of IDT in uterine strips of pregnant rats. Points represent mean ± SEM of six uterine strips. **P < 0.01.
preincubated with the COX inhibitor, indomethacin (INDO), exhibited a low stability in IDT, reaching at 40 min a decrement significantly greater than control (arOund 90%). PAF evoked sustained contractile activity with a lesser decay by 40 min as compared to control (Fig. 2) Uterine preincubations with L-NMMA or INDO diminished the effect on the contractions elicited by PAF (Fig. 3). Initial IDT values were similar in all the conditions tested (Table). © Harcourt Brace & Co Ltd 1998
The synthesis of PGE2 was stimulated by PAF. The effect was prevented when tissues had been pretreated with L-NMMA. PGF2 alpha production was not affected by PAF in any condition. There was no significant effect of L-NMMA preincubation on the basal synthesis of either PGE2 or PGF2 alpha (Fig. 5). DISCUSSION
Prostanoids and PAF are both uterotonic agents able to elicit myometrial contractions. 17 In an earlier work PAF had been proposed as a trigger of endogenous PG synthesis rather than a direct agonist on the myometrial contractions. 1
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 55-59
58
Chaud et al
2OO
E 8 loo
CONTROL
PAF
Fig. 4 Effect of PAF (10 ~M) on NOS activity measured by conversion of [~4C] L-citrulline in uterine strips of pregnant rats. Each column represents mean ± SEM of six uterine strips. ***P< 0.001.
m
PGF2alpha
m
PGE2
e~ .=o "6
CONTROL
PAF
L-NMMA PAF+L-NMMAPAF+D-NMMA
Fig. 5 Effect of PAF (10 ~M) on basal [~4C]-arachidonicacid in uterine strips of pregnant rats preincubated or not with L-NMMA or D-NMMA (300 I~M). Each bar represents mean + SEM of six uterine strips. **P< 0.01.
able regarding uterine tissue. This study shows that myometrial response to PAF was inhibited by L-NMMA, suggesting that NO might be involved in the ability of PAF to maintain a contractile effect. Moreover the present results demonstrate that NOS was not only present in the pregnant rat uterus at day 5, but it could be also increased by PAF. Accumulated evidence suggests that a paracrine interaction between PAF and PGs is essential in the implanting u t e r u s . 2a'24 It has been demonstrated that the addition of PAF to the glandular fraction of the endometrium causes an increase in PGE2 synthesis. H'12 We confirmed here this action but we also found that the enhancement of PGE2 induced by PAF was dependent on endogenous NO production. Therefore it appears that NO might be involved in the PC-mediated uterine contractile response to PAF. The iron-containing COX enzyme is a potential target for NO. 25 We had reported in an earlier work that NO is capable of stimulating PG production in the rat uterus) 6 There is also evidence indicating that agents that induce NO production such as lipopolysaccharide and interleukin-l[3 lead to the simultaneous release of products from the COX pathway. 26'27 In conclusion, our results indicate that NO could be a signal that mediates in the interaction between PAF and PGs and might represent an important mechanism modulating PAF actions. ACKNOWLEDGEMENTS
This work was supported by Grant PID-BID 0418 from the Consejo Nacional de Investigaciones Ciemfficas y T&nicas and Bag6 S. A. (Argentina). REFERENCES
Results presented here showed that PAF added exogenously was able to maintain uterine contractions with a reduced rate of decay during the period examined. This response was inhibited b y INDO, confirming its dependence on PG synthesis. Uterine spontaneous motility, known to depend on PGs, was also considerably impaired by INDO. On the contrary L-NMMA improved spontaneous myometrial activity which is in agreement with the known property of NO as a powerful myometrial relaxant. Relaxation is thought to be mediated by the increase of c-GMP which activates a c-GMP-dependent protein kinase leading to a reduction in intracellular Ca 2÷ concentrations, thus acting to cause a decrease in the force and frequency of contractions.19 There are some reports suggesting a role of NO in vasodilatation, vascular permeability and oedema formation in response to PAF, 2°-22 but there are no data avail-
1. Novaro V., Rettori V., Gonzalez E. et al. Interaction between uterine PGE and PGF2~production and the nitrixidergic system during embryonic implantation in the rat. Prostaglandins 1996; 51: 363-376. 2. Bredt D. S., Snyder S. H. Isolation of nitric oxide synthase, a calmodulin-required enzyme. Proc Natl Acad Sci USA 1990; 87: 682-687. 3. Minhas B. S., Zhu Y. P., Kim H. N., Burwinkel T. H., Ripps B. A., Buster J. E. Embryonic platelet activating factor production in the rabbit increases during the preimplantation phase. JAssist Reprod Genet 1993; 10: 366-370. 4. O'Neill C. Examination of the causes of early pregnancy associated thrombocytopenia in mice. JReprod Fertil 1985; 73: 567-577.
5. Spinks N. R., O'Neill C. O. Antagonists of embryo-derived platelet-activating factor prevent implantation of mouse embryos.JReprod Fertil 1988; 84: 89-98. 6. O'Neill C. O. Embryo-derived platelet activating factor: a preimplantation embryo mediator of maternal recognition of pregnancy. Domestic Animal EndocrinologF 1987; 4: 69-85. 7. Greep R. O., ed. Handbook of Physiology.Endocrine Control of
Prostaglandins, Leukotrienesand Essential Fatty Acids (1998) 58(I), 55-59
© Harcourt Brace & Co Ltd 1998
NO mediates PAF stimulatory action on uterine PG production
8.
9.
10.
11.
12. 13. 14.
15.
16.
17.
Egg Implantation. Baltimore MD USA: Williams and Wilkins, 1973: 187-215. Twafik O. W., Sagrillo C., Johnson D. C., Dey S. K. Decidualization in the rat: role of leukotrienes and prostaglandins. Prostaglandins Leukot Med 1987; 29:221-227. Phillips C. A., Poyser N. L. Studies on the involvement of prostaglandins in implantation in the rat. JReprod Ferl~'11981; 62: 73-81. Kudolo G. B., Kasamo M., Harper M. Autoradiographic localization of platelet-activating factor (PAF) binding sites in the rabbit endometrium during the peri-implantation period. Cell Tissue Res 1991; 231-241. Yee G. M., Squires P. M., Cejic S. S., Kennedy T. G. Lipid mediators of implantation and decidualization. JLipid Mediat 1993; 6: 525-534. Psychoyos A., Nikas G., Gravanis A. The role of prostaglandins in blastocyst implantation. Human Reprod 1995; 10: 30-42. Norman J. E., Cameron I. T. Nitric oxide in the human uterus. Reviews of ReproducEon 1996; 1: 61-68. Moncada S., Palmer R. M., Higgs E. A. Nitric oxide: physiology, pathophysiology and pharmacology. PtugrmacolRev 1991; 43: 109-142. Suburo A. M., Chaud M., Franchi A., Polak J. M., Gimeno M. F. Distribution of neuronal and non-neuronal NADPH diaphorases and nitric oxide synthases in rat uterine horns under differem hormonal conditions. Biol Reprod 1995; 52: 631-637. Franchi A., Chaud M., Rettori V., Suburo A., McCann S. M., Gimeno M. Role of nitric oxide in eicosanoid synthesis and uterine motility in estrogen-treated rat uterus. Proc Natl Acad Sci USA 1994; 91: 539-543. Carsten M. E., Miller J. D., eds. Uterine Function. Molecular and Cellular Aspects. New York and London: Plenum Press, 1990: 486-487.
© Harcourt Brace & Co L td 1998
59
18. Montrucchio G., Alloatti G., Tetta C., Roff'mello C., Emanuelli C., Camussi G. In vitro contractile effect of platelet-activating factor on guinea pig myometrium. Prostaglandins 1986; 32: 539-554. 19. Word A. R., Casey L., Kamm K. E., Stun J. T. Effects of cGMP on [Ca~+] myosin light chain phosphorylation and contraction in human myometrium. Am J Physiol 1991; 260:861-867. 20. Juncos L. A., Ren Y. L., Arma S., Ito S. Vasodilator and constrictor actions of platelet-activating factor in the isolated microperfused afferent arterile of the rabbit kidney. Role of endothelium derived relaxing factor/nitric oxide and cyclooxygenase products. JClin Invest 1993; 91: 1374-1379. 21. Texeira M. M., Willams T.J., Hellewell P. G. Role of prostaglandins and nitric oxide in acute inflammatory reactions in guinea pig skin. B r J P t u ~ o 1 1 9 9 3 ; 110: 1515-1521. 22. Filep J. G., Foldes-Filep E. Modulation by nitric oxide of platelet activating factor induced albumin extravasation in the conscious rat. BrJPharmaco11993; 110: 1347-1352. 23. Minhas B. S., Ripps B. A., Zhu Y. P., Kim H. N., Butwinkel T. H., Gleicher N. Platelet activation factor and conception. Am J ReFrod Immuno11996; ~$: 267-271. 24. Harper M.J. Platelet activating factor: a paracrine factor in preimplantation stages of reproduction? Bid Reprod 1989; 40: 907-913.
25. Salvemini D., Misko T. P., Masferrer J. L, Seibert K., Currie M. G., Needleman P. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci USA 1993; 90: 7240-7244. 26. Inoue T., Fukuo K., Morimoto S., Koh E., Ogiha T. Nitric oxide mediates interleukin-1 induced prostaglandin Ez production by vascular smooth muscle cells. Biochem Biophys Res Commun 1993; 194: 420-424. 27. Sautebin L., Ialenti A., Ianaro A., Di Rosa M. Modulation by nitric oxide of prostaglandin biosynthesis in the rat. BrJ Pharmacol 1995; 114: 323-328.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 55-59
Nitric oxide mediates platelet. activating factor stimulatory action on uterine prostaglandin production M. A. Chaud, A. M. Franchi, M. Ber6n de Astrada, M. F. Gimeno Centro de Estudios Farmacol6gicos y Botdnicos, Consejo Nacional de Investigaciones Cientificas y T~cnicas Serrano 669, 1414, Buenos Aires, Argentina
Accumulated evidence suggests that platelet-activating factor (PAF) may have a role in implantation by stimulating prostaglandin (PG) production. Since we had demonstrated that nitric oxide (NO) can increase uterine PGs, the aim of this study was to explore whether or not NO could mediate rat uterus responses to PAF on day 5 of gestation, when implantation takes place. Uterine motility was enhanced by PAF as compared to controls. This action was abolished by either the arginine analogue, N-monomethyl L-arginine (L-NMMA) or the cyclooxygenase inhibitor, indomethacin. On the other hand, NOS activity was detected in uterine strips and could be stimulated by PAF. The cyclooxygenase product PGE2 was also significantly stimulated by PAF. Inhibition of endogenous NO formation abolished the PAF effect on PG synthesis. Our results suggest that NO is an important intermediate in the interaction between PAF and PGs. Summary
INTRODUCTION
Increasing embryonic platelet-activating factor (PAF) production in the developing embryo prior to implantation might act as one of the initial signals that precedes the increase in vascular permeability and vasodflatation in the endometrium) -6 Local vasodilatation within the uterus is essential for the initiation of successful implantation, z Prostaglandins (PGs) have been proposed as the mediators of vascular permeability but also as required components of the progression of decidualization. 8,9 PAF binding sites are predominantly located in luminal epithelium 1° and it has been shown that PAF causes an increase in PG synthesis in the glandular fraction of the endometrium. 11,12 Nitric oxide (NO) is a gas molecule known to play an important role in many biological systems. Potential roles for NO on uterine function include vasodilatation and modulation of myometrial contractility, m4 We have demonstrated nerve fibers synthesizing NO in the uterus 15and showed that uterine NO has complex opposReceived 8 October 1996 Accepted 12 May 1997 Correspondence to: M. F. Gimeno, Fax. 00 54 01 856 2751
ing actions, relaxing uterine muscle by cGMP and contracting it via stimulation of the cyclooxygenase (COX) pathway, producing various prostanoids. 16 The present study was undertaken to examine a possible role of NO as a mediator of PAF actions in the rat uterus at day 5 of gestation. MATERIALS AND M E T H O D S Animals
Female rats of the Wistar strain (200-230 g body weight) were used. They were housed in group cages under controlled conditions of light (lights on 05:00-17:00 h) and temperature (23-25°C). Rat chow and water were freely available. Mating was confirmed by the presence of sperm in vaginal smears the next moming and this was considered to be the first day of gestation. Uterine tissue was obtained from rats at day 5 of gestation. Animals were killed by a blow on the neck and their uterine horns were removed. Fetuses, placenta and fetal membranes were separated and discarded. Metabolism of
[14C] arachidonic acid
Once the uterine horns were obtained, each horn was opened, trimmed of visible fat, and placed in a Petri dish 55
56
Chaud et al
containing a modified Krebs-Ringer bicarbonate (KRB) solution with glucose (11 raM). 1 The metabolism of exogenous arachidonic acid by rat uterine tissue was determined by incubating the tissue for 60 rain in KRB medium containing 0.25 ~tCi of [1'C] arachidonic acid (55 mCi/mol) in an atmosphere of 95% O2/CO2 with constant shaking at 60 cycles per rain at 37"C. For each determination, 200 nag of uterine tissue was used. The uterine strips were randomly treated with PAF (0.1 laM) or NG-monomethyl-L-arginine (NMMA) (300 glVl). These reagents were added to the incubation medium alone or in combination as described in the Results. The controls were incubated in medium alone. At the end of the incubation period the incubation medium was acidified to pH 3.0 with 1.0 M HCI in 1 vol of ethyl acetate and extracted twice for PGs. Pooled ethyl acetate extracts were dried under nitrogen. The residues were suspended in chloroform/methanol and applied to silica gel TIC plates. The plates were developed in a solvent system of benzene/ dioxane/glacial acetic acid (60:30:3; vol/vol). The position of the authentic eicosanoids was visualized by spraying the dried plates with 10% phosphomolybdic acid in ethanol followed by heating at 110°C for 10 min. Average Rf values were 0.35 for PGF2~, 0.47 for PGE2 and 0.8 for arachidonic acid. Radioactivity from TLC zones for arachidonic acid and for different prostanoids was measured by liquid scintillation counting. The area of each of the radioactive peaks corresponding to authentic prostanoids was calculated and expressed as a percentage of the total radioactivity of the plates.
NOS enzyme activity was quantified by a modified method of Bredt and Snyder, 1 which measures the conversion of [14C]-arginine into [14C)-citrulline. Slices were incubated at 37°C in a buffer that contained 20 nM HEPES, 10 ~VI [14C]-arginine (0.3 ~tCi) and 0.5 mn NADPH. After 15 min of incubation, samples were homogenized using a Tissuemizer. The samples were centrifuged for 10 minutes at 10 000 rpm and then they were applied to a 1 ml DOWEX AG5OW-X* column (Na+-form) and [~4C]citruUine was eluted in 3 ml of water. The radioactivity was measured by liquid scintillation counting. Enzyme activity is reported in cpm citrulline/1 O0 mg wet weight.
Motility studies
Statistics
Uterine horns were obtained as described above. The tissue was removed and each horn was divided by a transverse cut into two equal-length segments. The segments were placed in Petri dishes containing KRB at room temperature and constantly gassed with 95% OJCO2. Each segment was immediately opened by a cut along the mesosalpinx insertion; one end was attached to a glass holder and immersed in a tissue chamber filled with 20 ml of KRB (pH 7.4 37°C) and continuously gassed. The other end of the tissue was attached to a strain gauge coupled to an amplifier driving a direct writing oscillograph. After a resting tension of 1 g was applied to each strip by means of a micrometric device, isometric developed tension (IDT) and frequency of contractions (FC) were measured. IDT values (expressed in nag) were obtained by measuring the mean amplitude of all the contractions recorded over a 10-min period. FC values were obtained as the mean number of the contractile cycles observed during the same period. All uterine strips were preincubated in the chambers without tension, in the presence of NMMA (300 pM), indomethacin (1 pM) or
All values presented in this study represent means + SEM. Comparisons between groups were performed employing the ANOVA test. Differences between means were considered significant when Pwas 0.05 or less.
in an equal volume of medium alone for 30 min (control). After uteri preincubation a single dose of PAF (0.1 ~VI) was added. Uterine contractions in the first 10 min were taken as control. IDT and FC variations during the incubation period were expressed as a percentage change from the control. NOS enzyme assay
Drugs and chemicals
[1-14C]-arachidonic acid was provided by New England Nuclear (specific activity 56 ~tCi/mmol); L-[U14C]-arginine monohydrochloride was purchased from Amersham (specific activity 317 mCi/mmol); DOWEX AG5) W-X8 column (Na form) was provided by Bio Rad. All other reagents were obtained from Sigma Chemical Company.
RESULTS Effect of L-NMMA or indomethacin on PAF-induced motility
Figures 1-3 show the variations in uterine spontaneous motility during 40 min incubation. Data were expressed as percentage of change from the initial IDT (see Material and methods). FC values were also evaluated. Since the results obtained in FC followed the same patterns as IDT these data were not shown. As can be seen in Figure 1, the IDT of control uterine strips had an appreciable decay over time (60% after 40 min incubation). Uterine strips preincubated with the NOS inhibitor, L-NMMA showed considerably less change over 40 rain (30% decay after 40 roan). Tissues
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 55-59
© Harcourt Brace & Co Ltd 1998
NO mediates PAF stimu/atory action on uterine PG production
o PAF
= CONTROL Q NIIIt1~
0'
c-
"
a
0
" 1141)0
-20.
57
-G =
rtl111A+Pgr
, ]lll)0+PIg
-ZO.
c
-40.
~'
-40"
o
-60.
o
-6D~
#__
-80.
'-'
-I00
0
10
~0 ,~0 l]14E(m]n)
-80. -1OD 0
40
Fig. 1 Effect of 30 min preincubation with L-NMMA (300 I~M) or INDO (1 I~M) on the time course of IDT in uterine strips of pregnant rats. Points represent mean ± SEM of six uterine strips. **P < 0.01, *P < 0.05.
• CONIR0t o PAF
10
~'0 30 lillE(mini
40
Fig. 3 Effect of 30 min preincubation with L-NMMA (300 IIM) or INDO (1 I~M) on the time course of PAF (10 ~M) on the time course of IDT in uterine strips of pregnant rats. Points represent mean ± SEM of six uterine strips. **P < 0.01.
Table Effect of 30 min preincubation with L-NMMA (300 IIM) or INDO (100 llM) on initial IDT induced by PAF (10 llM) Initial IDT
-20.
Control INDO NMMA
~J cos cf=J
-40.
42 + 5 (6) 37 ± 4 (6) 28 ± 4 (6)
NOS activity was assessed in uterine strips by measuring the conversion of L-arginine to L-citrulline. As shown in Figure 4, uterine NOS activity was detected in 5 day pregnant rats and could be enhanced by PAF.
I--
-80]. 0
PAF INDO + PAF NMMA + PAF
Effect of PAF on uterine N O S activity
-60.
-100
30 + 4 (6) 35 + 3 (6) 35 ± 6 (6)
Ib
i0
4b
Effect of L - N M M A on the production of PGs from labelled A A in response to PAF
l]ME(miB) Fig. 2 Effect of PAF (10 ~M) on the time course of IDT in uterine strips of pregnant rats. Points represent mean ± SEM of six uterine strips. **P < 0.01.
preincubated with the COX inhibitor, indomethacin (INDO), exhibited a low stability in IDT, reaching at 40 min a decrement significantly greater than control (arOund 90%). PAF evoked sustained contractile activity with a lesser decay by 40 min as compared to control (Fig. 2) Uterine preincubations with L-NMMA or INDO diminished the effect on the contractions elicited by PAF (Fig. 3). Initial IDT values were similar in all the conditions tested (Table). © Harcourt Brace & Co Ltd 1998
The synthesis of PGE2 was stimulated by PAF. The effect was prevented when tissues had been pretreated with L-NMMA. PGF2 alpha production was not affected by PAF in any condition. There was no significant effect of L-NMMA preincubation on the basal synthesis of either PGE2 or PGF2 alpha (Fig. 5). DISCUSSION
Prostanoids and PAF are both uterotonic agents able to elicit myometrial contractions. 17 In an earlier work PAF had been proposed as a trigger of endogenous PG synthesis rather than a direct agonist on the myometrial contractions. 1
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 55-59
58
Chaud et al
2OO
E 8 loo
CONTROL
PAF
Fig. 4 Effect of PAF (10 ~M) on NOS activity measured by conversion of [~4C] L-citrulline in uterine strips of pregnant rats. Each column represents mean ± SEM of six uterine strips. ***P< 0.001.
m
PGF2alpha
m
PGE2
e~ .=o "6
CONTROL
PAF
L-NMMA PAF+L-NMMAPAF+D-NMMA
Fig. 5 Effect of PAF (10 ~M) on basal [~4C]-arachidonicacid in uterine strips of pregnant rats preincubated or not with L-NMMA or D-NMMA (300 I~M). Each bar represents mean + SEM of six uterine strips. **P< 0.01.
able regarding uterine tissue. This study shows that myometrial response to PAF was inhibited by L-NMMA, suggesting that NO might be involved in the ability of PAF to maintain a contractile effect. Moreover the present results demonstrate that NOS was not only present in the pregnant rat uterus at day 5, but it could be also increased by PAF. Accumulated evidence suggests that a paracrine interaction between PAF and PGs is essential in the implanting u t e r u s . 2a'24 It has been demonstrated that the addition of PAF to the glandular fraction of the endometrium causes an increase in PGE2 synthesis. H'12 We confirmed here this action but we also found that the enhancement of PGE2 induced by PAF was dependent on endogenous NO production. Therefore it appears that NO might be involved in the PC-mediated uterine contractile response to PAF. The iron-containing COX enzyme is a potential target for NO. 25 We had reported in an earlier work that NO is capable of stimulating PG production in the rat uterus) 6 There is also evidence indicating that agents that induce NO production such as lipopolysaccharide and interleukin-l[3 lead to the simultaneous release of products from the COX pathway. 26'27 In conclusion, our results indicate that NO could be a signal that mediates in the interaction between PAF and PGs and might represent an important mechanism modulating PAF actions. ACKNOWLEDGEMENTS
This work was supported by Grant PID-BID 0418 from the Consejo Nacional de Investigaciones Ciemfficas y T&nicas and Bag6 S. A. (Argentina). REFERENCES
Results presented here showed that PAF added exogenously was able to maintain uterine contractions with a reduced rate of decay during the period examined. This response was inhibited b y INDO, confirming its dependence on PG synthesis. Uterine spontaneous motility, known to depend on PGs, was also considerably impaired by INDO. On the contrary L-NMMA improved spontaneous myometrial activity which is in agreement with the known property of NO as a powerful myometrial relaxant. Relaxation is thought to be mediated by the increase of c-GMP which activates a c-GMP-dependent protein kinase leading to a reduction in intracellular Ca 2÷ concentrations, thus acting to cause a decrease in the force and frequency of contractions.19 There are some reports suggesting a role of NO in vasodilatation, vascular permeability and oedema formation in response to PAF, 2°-22 but there are no data avail-
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