Acta Physiol Scand 1992, 146, 129-134
Influence of prostaglandins and a prostaglandin H synthase inhibitor on cervical secretion of the guinea-pig B. JONSSON" and M. H A M M A R S T R 0 M " t
* Department of Obstetrics and Gynecology, Karolinska Hospital, Karolinska Institute, Sweden and Department of Physiology I, Karolinska Institute, Sweden
t
JONSSON, B. & HAMMARSTROM, M. 1992. Influence of prostaglandins and
a prostaglandin
H synthase inhibitor on cervical secretion of the guinea-pig. Acta Physiol Scand 146, 129-134. Received 2 April 1991, accepted 17 March 1992. ISSN 0001-6772. Department of Obstetrics and Gynecology, Karolinska Institute, Sweden. The modulatory effects of several prostaglandins and a prostaglandin H synthase inhibitor (indomethacin) on basal as well as nerve stimulation induced secretion from the cervical glands of the guinea-pig were studied. Hypogastric nerve stimulation resulted in a secretory response of 113%. Indomethacin dose dependently inhibited this secretory response. Prostaglandins E,, F,, and I, inhibited and 19-OH prostaglandin El reduced nerve stimulation induced secretion. Prostaglandin I, and 19-OH PGE, markedly enhanced basal secretion, while indomethacin as well as PGE, and PGF,, did not induce any secretion of cervical glands. However, PGFZain combination with the aadrenergic blocker phentolamine resulted in an increase in secretion. The inhibitory effect of prostaglandins on cholinergic secretory innervation might be due to stimulation of adrenergic nerves exerting an inhibitory influence on cholinergic secretomotor innervation. It is suggested that PGI, and 19-OH PGE, exert postjunctional stimulatory effects on the secretory lining and that a lack of secretory effect of PGF,, at least in part may be due to stimulatory effects on adrenergic neurons, inhibiting cholinergic secretomotor transmission. Thus, in this in vitro study it is shown that metabolites of the arachidonic acid cascade and a prostaglandin H synthase inhibitor can modulate cervical secretion and thus maybe influence fertilization.
+
Key words: autonomic nerves, cervical secretion, cervix uteri, cholinergic, prostaglandins, prostaglandin H synthase inhibitors. Cervical gland secretion is important for mammalian reproduction, e.g. sperm transport, survival and capacitation. The cervical mucus is well known to display characteristic cyclic variations. At ovulation time the secretory products become abundant, watery and viscous and the mucin component is formed as long fibrils (Odeblad 1973, ChrQien 1974, Chang & Hunter 1975). In the guinea-pig the cervical glands receive a nervous supply via the hypogastric nerve. Neurons relay in the paracervical ganglion and Correspondence : Margareta Hammarstrom, Department of Obstetrics and Gynecology, Karolinska Hospital, S-104 01 Stockholm, Sweden.
the postganglionic neurons are cholinergic histochemical (Hammarstrom 1989). By methods, acetylcholinesterase positive nerves have been shown to supply cervical glands of the guinea-pig and human uterus, and adrenergic nerves have been demonstrated in connection with the cervical mucosa (Adam & Schenk 1969, Hammarstrom & Sjostrand 1980, Lakomy et al. 1982). Uterine tissue has the capacity to synthesize prostaglandins which play a prominent role in the regulation of different uterine functions (Christensen 1984), and also modulate autonomic neurotransmission (Gustafsson 1989). Semen of several species, e.g. human, sheep and monkey, is rich in prostaglandins (Eliasson 1959, Moghissi
129 F
ACT 146
130
B. Jonsson and M . Hammnsstsom 7
T
/I
6'
I 107106
io5 M
10" M
id
i o 6 lo5 M
id
lo6 l o 5 M
1
Fig. 1. Effects of prostaglandins on hypogastric nerve stimulation (. Hz, i 30 V, 0.1 ms) at ovulation time. Hypogastric nerie stirnulation [controls (CJ)] increased secretion by 113y0 from resting secretion level. PGI, .( 10-'-10F xi) depressed the secretor!. response and 19-OH PGE, (@j 10.' 11) reduced nerve stimulation induced secretion ("P < 0.05 vs. controls and prostaglandin I,, F22 and EJ. PGE, (m 10-'-10-" xf) as well as PGF2x(B 10-'-10-" 34) abolished the nerve M) was not reversed by stimulation induced secretion. The inhibitory effect of PGFZz( combination with phentolamine ([III] 1 0 F xi). (\lean and SEM; number of experiments indicated h!- figures at columns.)
8i LIurraJ- 1970, Gottlieb 1988) and especiallJ- in IC)-OH PGE, (Taylor 8i Kelly 1974), a n d endometrial a n d ceri~ical secretion contains prostaglandin E, and F,, (Charbonnel r t (11. 1982). T h u s , a n attempt w s made to elucidate possible effects of prostaglandins and a prostaglandin H s!-nthase inhibitor o n cervical secretion in a guinea-pig model in r i m .
m ' r INID s A4nrniu/s. Mottled or albino virgin guinea-pigs (35(t500 g) were used. T h e animals were controlled daily concerning the status of the vaginal closure membrane. Only animals showing regular cycles were used. .Wer two normal cycles, a smear was taken when the membrane nas open. T h e day of maximum cornification before leucocyte influx was designated da! 1 (Stockard 8, Papanicolaou 1917, 1919, Bland 8, D o n o n n 1970).Experiments were undertaken around ovulation da!- 16 or da!- 1. Dissrc ring iind mounting procedures. Animals \\ere stunned and bled. ?'he uterus, cerkix and upper parts of the Lagina were remoi-ed together with the -
hgogastric nerve, carefully dissected. T h e uterine horns and parametrical tissues were removed. T h e cenix was diJ-ided longitudinally and each piece was tied with the mucosal side out along a stiff polyethylene catheter and then placed in organ baths. T h e adjacent nerves were placed within the bath on platinum electrodes ( 2 mm apart) at a distance of 15 mm from the cervis. -Yrrw stitnulation was applied with 5 Hz, 30 V, 0.1 ms for 10 s every 30th second during the sampling periods. Experinrental prtjorrnanre. Experiments were performed following a modified schedule given by Hammarstrom (1989). T h e organs were accommodated in Tyrode solution (36 "C) changed every 20 min during 1 h for equilibration. Sampling was then started and included six periods of 10 min in Tyrode solution. For determination of basal secretion levels the mean of periods 1, 2 , 1 and 6 was used. During periods 3 and 5 field stimulation of nerves and/or administration of drugs mere performed. Secretory responses vvere calculated as percent change from basal secretion level. Diu/ysing procedure. The collected fluid (25 ml of each period) was then dialysed (cut-off' 6000-
Prostaglandins injuence cervix secretion 7
131
T
6
T
1
-7
LO
M
-6
io
105
M
Fig. 2. Effects of a prostaglandin H synthase inhibitor (indomethacin) on resting secretion level as well as on nerve stimulation ( 5 Hz, 30 V, 0.1 ms) induced secretion at ovulation time. increased secretion by 113%. Indomethacin (m Hypogastric nerve stimulation [controls (O)] 10-7-10-5 M) dose-dependently inhibited this nerve stimulation induced secretion. However, indomethacin (m 10-7-105 M) did not statistically significantly influence resting secretion level. (Mean and SEM; number of experiments indicated by figures at columns.)
8000 MW Spectrapor Membrane). Distilled water (M "C) was used and changed after 6, 12, 24 and 36 h (total time 48 h). Estimation of secretion. The lyophilized samples were redissolved in 3 ml of 14.4 M H,SO,. 0.1 ml 8.5 x M indole was added, the sample taken and then heated (100 "C) for 10 min. The samples were read at 470 nm on a Zeiss spectrophotometer. The amounts of cervical secretion were arbitrarily quantified and calculated as glucose/galactose, using an internal standard (Hammarstrom 1980, 1989). Basal secretion level varied from experiment to experiment between M glucose/galactose per 10 min. 0.1 and 1 x Calculation and statistics. Dunnett's test for multiple comparison was used for statistical evaluation of experiments (Kirk 1968). Tyrode solution was composed of NaCl 13.7 x lo-', KCI 2.7 x MgCl, 4.9 x lo-*, CaCI, 1.8 x NaHCO, 1.2 x lo-' and Na,HPO, 2H,O 3.1 x mol I-'. 1.1 x lo-' mol of glucose was added per litre. Drugs. Phentolamine hydrochloride (Ciba, Basel, Switzerland), prostaglandin E, (Sigma, St Louis, MO, USA), prostaglandin FZa(Sigma), prostaglandin I, (Sigma), 19(R)-hydroxy prostaglandin E, (Cayman
Chemical, Ann Arbor, MI, USA) and indomethacin (Dumex, Copenhagen, Denmark) were used.
RESULTS Nerve stimulation Hypogastric nerve stimulation increased secre10; n = 7) (Figs 1, 2 & 3). tion by 113% (i-
Drug efects on hypogastric nerve stirnulation induced secretion (Figs 1 6 2) Prostaglandin I, ( 10P7-10-' M) as well as prostaglandin E, ( 10-7-10-5 M) prominently inhibited nerve stimulation induced secretion (P < 0.05). Prostaglandin F,, ( 10-7-10-5 M) also markedly depressed nerve induced secretion (P< 0.05), and 19-OH PGE, (loP6M) reduced secretory responses to a level of 31 yo(k6 ; n = 6) ( P < 0.05 vs. controls and other prostaglandins). Phentolamine (loP6 M) did not reverse the inhibitory effect of PGF,, (lo-' M) on hypo-
+
5-2
132
B.Jonsson and M . Hummarstrom 7
J Fig. 3. Effects of prostaglandins on resting secretion level from cervical glands at ovulation time. Hypogastric nerve stimulation [controls increased secretion by 113?b. Prostaglandin I, (m 10." M) enhanced secretion to the same extent as did nerve stimulation (n.s. vs. controls, P < 0.05 vs. PGE, and PGFJ. Also 19-OH PGE, ( @ M) had a marked stimulatory effect on secretory linings (P < 0.05 s's. controls, PGE, and PGF,J. Prostaglandin E, (@ 10-7-10-5 M) as well as PGF,, (g 10-'-10-' M) did not induce an>- carbohydrate secretion. Administration of the adrenergic blocker phentolamine X)I in combination with PGF,, (m) resulted in a marked secretory response ( P < 0.0-5 s's. PGF,z; 10-'-10-" 51). (Mean and SER.1; number of experiments indicated by figures a t columns.)
(o)]
gastric nerve stimulation ( - 1 0 1 4 6 ; IZ = 4) (Fig. 1). Indomethacin (10-'-10-' hi) (Fig. 2 ) dose-dependent11 inhibited the nerve stimulation induced secretion ( P < 0.05).
Drug ejects on basal secretion (Figs2 6 3) Prostaglandin I, resulted in a dose dependent secretory response, and lo-' bi PGI, increased secretion to the same extent as did nerve stimulation ( 76", i 18; n = 4). 19-OH PGE, (10- li M) also resulted in an increase of secretion (+.5joo& 18; n = 6 ; P < 0.05 vs. controls and PGE:,, PGF,,). Prostaglandin E, ( 10-7-10-~5\I) and PGF,, (10-'-10-' M) had no effects on cervical secretion ( P < 0.05). T h e combination M) and PGF,, ( M) of phentolamine ( resulted in a secretory response of 4000 ( f 18; ti = 6 ; P < 0.05 vs. controls and PGF,, M) (Fig. 3 ) . Indomethacin (10-7-10-' M) did not y-ield any carbohydrate secretion (Fig. 2 ) .
+
+
DISCUSSION Prostaglandins, known potent neuromodulators of autonomic nervous transmission, are synthesized in and exert different and contradictory effects on the uterus. I n the human a high concentration of prostaglandins are present as a result of the seminal fluid deposed close to the uterus. Semen of several species as e.g. the guinea-pig is considered to contain low levels of prostaglandins (Eliasson 1959, Cenedella 1975). Even small amounts of prostaglandins are, however, supposed to exert biological effects (Cenedella 1975). Despite methodological obstacles it was supposed to be of interest to study the effects of prostaglandins and a prostaglandin H synthase inhibitor on cervical secretion, which is supposed to be of great importance for fertilization. An enhancement of cholinergic neurotransmission after application of prostaglandins has
Prostaglandins injuence cervix secretion been shown (Gustafsson 1989). Furthermore, PGF,, has been shown to exert secretory effects on salivary glands (Hahn & Patil 1972). In this study, application of 19-OH PGE, resulted in an apparent secretory response. Also PGI, induced a secretory response. The effect is probably postjunctional as nerve stimulation in combination with these prostaglandins does not result in any prominent secretory effect. Furthermore, prejunctional stimulation of cholinergic neurons is rarely demonstrated (Gustafsson 1989). Also Hahn & Patil (1974) suggest a postjunctional secretory effect by PGF,, since tetrodotoxin does not reduce the secretory response induced in salivary glands. Prostaglandin E, does not have any secretory effects and this is in line with findings of PGE, on gastric secretion (Sautereau et al. 1989). All prostaglandins except 19-OH PGE, abolished nerve stimulation induced secretion. The inhibitory effect of prostaglandins on nerve stimulation induced secretion might represent stimulation of adrenergic nerves thus unmasking an adrenergic inhibition. In the endometrium of the guinea-pig an a-adrenergic inhibitory innervation has been shown although no adrenergic influence on nerve stimulation induced secretion from the cervical glands has been demonstrated (Hammarstrom 1985, 1989). The adrenergic inhibition of cholinergic secretomotor innervation of the endometrium was proven after postganglionic nerve stimulation and in studies on cervical secretion (Hammarstrom 1989) nerve stimulation was performed on isolated nerve preparations. Lack of demonstrable adrenergic inhibition in this hypogastric nerve-cervix uteri preparation might be due to the fact that nerve stimulation was mainly performed preganglionic and thus resulted in divergence in the synaptic region. The adrenergic inhibition might therefore be difficult to demonstrate. Concomitantly, the secretory responses to PGI, and PGF,, could be due to inhibition of adrenergic transmission, since secretory response to PGF,, is much enhanced after administration of the a-adrenergic inhibitor phentolamine. Prostaglandin I, and F,, primarily have adrenergic stimulatory effects (Gustafsson 1989). Indomethacin did not exert any secretory effects on cervical glands and further dosedependently inhibited nerve induced secretion. Indomethacin has been discussed as a potent calcium antagonist and it is well settled that
133
indomethacin effects could not to a full extent be reversed by prostaglandin application (Gustafsson 1989). The many pitfalls in studying prostaglandin modulation effects on autonomic neurotransmission is extensively reviewed by Gustafsson (1989). In this preparation there is also the complex peripheral autonomic nervous supply to the paracervical ganglia and uterus described in the review of Papka et al. (1987). Furthermore, metabolism of prostaglandins in this in vitro preparation could not be excluded (Christensen 1984). In conclusion, PGI, and 19-OH PGE, exert effects on basal secretion from cervical glands and seem to be potent postjunctional stimulators. Marked reduction of nerve stimulation induced secretion is seen after all prostaglandins, except after 19-OH PGE, which to a lesser extent reduced secretory responses. Indomethacin, frequently used in pharmacological doses as pain relief among fertile women, is a potent inhibitor of secretion induced by nerve stimulation. Thus, in this in vitro study metabolites of the arachidonic acid cascade and its inhibitor can modulate cervical secretion and thus maybe influence fertilization. The present study was supported by grants from the Karolinska Institute and the Swedish Medical Society. For excellent technical assistance we thank Ms Astrid
Haggblad.
REFERENCES ADHAM,N. & SCHENK,E.A. 1969. Autonomic innervation of the rat vagina, cervix and uterus and its cyclic variation. A m 3 Obstet Gynecol 104, 508-516. BLAND,K.P. & DONOVAN, B.T. 1970. Oestrogen and progesterone and the function of the corpora lutea in the guinea-pig. 3 Endocrinol47, 225-230. CENEDELLA, R.J. 1975. Prostaglandins and male reproductive physiology. In: J.A. Thomas & R.L. Singhal (eds.) Molecular Mechanisms of Gonadal Hormone Action, pp. 325-358. University Park Press, Baltimore. CHANG, M.C. & HUNTER, R.H.F. 1975. Capacitation of mammalian sperm : Biological and experimental aspects. In: D.W. Hamilton & R.O. Greep (eds.) Handbook of Physiology, Section 7, Endocrinology, vol. v, Male Reproductive System, pp. 339-351. American Physiological Society, Washington.
134
B. Jonsson and M . Hammarstrom
L, B., KREMER,31.. GEROZISSIS, K. & DR.;ZG, F. 1982. Human cervical mucus contains large amounts of prostaglandins. Ferril Steril 38, 109 - 111. CHRETIES,F.C. 1974. La glaire cervicale 111. Roles physiologiques. 3 (=)~nerolObsret Biol. Reprod 6, 481--488. CHRISTENSEN, N.J. 1984. Studies on the bioconversion ofarachidonic acid in human pregnant reproductive tissue. Thesis. Karolinska Instituter, Stockholm, Sweden, ISBN 91-5222-7389. EI.IASSON,R. 1959. Studies on prostaglandins. Occurrence, formation and biological actions.
Influence of prostaglandins and a prostaglandin H synthase inhibitor on cervical secretion of the guinea-pig B. JONSSON" and M. H A M M A R S T R 0 M " t
* Department of Obstetrics and Gynecology, Karolinska Hospital, Karolinska Institute, Sweden and Department of Physiology I, Karolinska Institute, Sweden
t
JONSSON, B. & HAMMARSTROM, M. 1992. Influence of prostaglandins and
a prostaglandin
H synthase inhibitor on cervical secretion of the guinea-pig. Acta Physiol Scand 146, 129-134. Received 2 April 1991, accepted 17 March 1992. ISSN 0001-6772. Department of Obstetrics and Gynecology, Karolinska Institute, Sweden. The modulatory effects of several prostaglandins and a prostaglandin H synthase inhibitor (indomethacin) on basal as well as nerve stimulation induced secretion from the cervical glands of the guinea-pig were studied. Hypogastric nerve stimulation resulted in a secretory response of 113%. Indomethacin dose dependently inhibited this secretory response. Prostaglandins E,, F,, and I, inhibited and 19-OH prostaglandin El reduced nerve stimulation induced secretion. Prostaglandin I, and 19-OH PGE, markedly enhanced basal secretion, while indomethacin as well as PGE, and PGF,, did not induce any secretion of cervical glands. However, PGFZain combination with the aadrenergic blocker phentolamine resulted in an increase in secretion. The inhibitory effect of prostaglandins on cholinergic secretory innervation might be due to stimulation of adrenergic nerves exerting an inhibitory influence on cholinergic secretomotor innervation. It is suggested that PGI, and 19-OH PGE, exert postjunctional stimulatory effects on the secretory lining and that a lack of secretory effect of PGF,, at least in part may be due to stimulatory effects on adrenergic neurons, inhibiting cholinergic secretomotor transmission. Thus, in this in vitro study it is shown that metabolites of the arachidonic acid cascade and a prostaglandin H synthase inhibitor can modulate cervical secretion and thus maybe influence fertilization.
+
Key words: autonomic nerves, cervical secretion, cervix uteri, cholinergic, prostaglandins, prostaglandin H synthase inhibitors. Cervical gland secretion is important for mammalian reproduction, e.g. sperm transport, survival and capacitation. The cervical mucus is well known to display characteristic cyclic variations. At ovulation time the secretory products become abundant, watery and viscous and the mucin component is formed as long fibrils (Odeblad 1973, ChrQien 1974, Chang & Hunter 1975). In the guinea-pig the cervical glands receive a nervous supply via the hypogastric nerve. Neurons relay in the paracervical ganglion and Correspondence : Margareta Hammarstrom, Department of Obstetrics and Gynecology, Karolinska Hospital, S-104 01 Stockholm, Sweden.
the postganglionic neurons are cholinergic histochemical (Hammarstrom 1989). By methods, acetylcholinesterase positive nerves have been shown to supply cervical glands of the guinea-pig and human uterus, and adrenergic nerves have been demonstrated in connection with the cervical mucosa (Adam & Schenk 1969, Hammarstrom & Sjostrand 1980, Lakomy et al. 1982). Uterine tissue has the capacity to synthesize prostaglandins which play a prominent role in the regulation of different uterine functions (Christensen 1984), and also modulate autonomic neurotransmission (Gustafsson 1989). Semen of several species, e.g. human, sheep and monkey, is rich in prostaglandins (Eliasson 1959, Moghissi
129 F
ACT 146
130
B. Jonsson and M . Hammnsstsom 7
T
/I
6'
I 107106
io5 M
10" M
id
i o 6 lo5 M
id
lo6 l o 5 M
1
Fig. 1. Effects of prostaglandins on hypogastric nerve stimulation (. Hz, i 30 V, 0.1 ms) at ovulation time. Hypogastric nerie stirnulation [controls (CJ)] increased secretion by 113y0 from resting secretion level. PGI, .( 10-'-10F xi) depressed the secretor!. response and 19-OH PGE, (@j 10.' 11) reduced nerve stimulation induced secretion ("P < 0.05 vs. controls and prostaglandin I,, F22 and EJ. PGE, (m 10-'-10-" xf) as well as PGF2x(B 10-'-10-" 34) abolished the nerve M) was not reversed by stimulation induced secretion. The inhibitory effect of PGFZz( combination with phentolamine ([III] 1 0 F xi). (\lean and SEM; number of experiments indicated h!- figures at columns.)
8i LIurraJ- 1970, Gottlieb 1988) and especiallJ- in IC)-OH PGE, (Taylor 8i Kelly 1974), a n d endometrial a n d ceri~ical secretion contains prostaglandin E, and F,, (Charbonnel r t (11. 1982). T h u s , a n attempt w s made to elucidate possible effects of prostaglandins and a prostaglandin H s!-nthase inhibitor o n cervical secretion in a guinea-pig model in r i m .
m ' r INID s A4nrniu/s. Mottled or albino virgin guinea-pigs (35(t500 g) were used. T h e animals were controlled daily concerning the status of the vaginal closure membrane. Only animals showing regular cycles were used. .Wer two normal cycles, a smear was taken when the membrane nas open. T h e day of maximum cornification before leucocyte influx was designated da! 1 (Stockard 8, Papanicolaou 1917, 1919, Bland 8, D o n o n n 1970).Experiments were undertaken around ovulation da!- 16 or da!- 1. Dissrc ring iind mounting procedures. Animals \\ere stunned and bled. ?'he uterus, cerkix and upper parts of the Lagina were remoi-ed together with the -
hgogastric nerve, carefully dissected. T h e uterine horns and parametrical tissues were removed. T h e cenix was diJ-ided longitudinally and each piece was tied with the mucosal side out along a stiff polyethylene catheter and then placed in organ baths. T h e adjacent nerves were placed within the bath on platinum electrodes ( 2 mm apart) at a distance of 15 mm from the cervis. -Yrrw stitnulation was applied with 5 Hz, 30 V, 0.1 ms for 10 s every 30th second during the sampling periods. Experinrental prtjorrnanre. Experiments were performed following a modified schedule given by Hammarstrom (1989). T h e organs were accommodated in Tyrode solution (36 "C) changed every 20 min during 1 h for equilibration. Sampling was then started and included six periods of 10 min in Tyrode solution. For determination of basal secretion levels the mean of periods 1, 2 , 1 and 6 was used. During periods 3 and 5 field stimulation of nerves and/or administration of drugs mere performed. Secretory responses vvere calculated as percent change from basal secretion level. Diu/ysing procedure. The collected fluid (25 ml of each period) was then dialysed (cut-off' 6000-
Prostaglandins injuence cervix secretion 7
131
T
6
T
1
-7
LO
M
-6
io
105
M
Fig. 2. Effects of a prostaglandin H synthase inhibitor (indomethacin) on resting secretion level as well as on nerve stimulation ( 5 Hz, 30 V, 0.1 ms) induced secretion at ovulation time. increased secretion by 113%. Indomethacin (m Hypogastric nerve stimulation [controls (O)] 10-7-10-5 M) dose-dependently inhibited this nerve stimulation induced secretion. However, indomethacin (m 10-7-105 M) did not statistically significantly influence resting secretion level. (Mean and SEM; number of experiments indicated by figures at columns.)
8000 MW Spectrapor Membrane). Distilled water (M "C) was used and changed after 6, 12, 24 and 36 h (total time 48 h). Estimation of secretion. The lyophilized samples were redissolved in 3 ml of 14.4 M H,SO,. 0.1 ml 8.5 x M indole was added, the sample taken and then heated (100 "C) for 10 min. The samples were read at 470 nm on a Zeiss spectrophotometer. The amounts of cervical secretion were arbitrarily quantified and calculated as glucose/galactose, using an internal standard (Hammarstrom 1980, 1989). Basal secretion level varied from experiment to experiment between M glucose/galactose per 10 min. 0.1 and 1 x Calculation and statistics. Dunnett's test for multiple comparison was used for statistical evaluation of experiments (Kirk 1968). Tyrode solution was composed of NaCl 13.7 x lo-', KCI 2.7 x MgCl, 4.9 x lo-*, CaCI, 1.8 x NaHCO, 1.2 x lo-' and Na,HPO, 2H,O 3.1 x mol I-'. 1.1 x lo-' mol of glucose was added per litre. Drugs. Phentolamine hydrochloride (Ciba, Basel, Switzerland), prostaglandin E, (Sigma, St Louis, MO, USA), prostaglandin FZa(Sigma), prostaglandin I, (Sigma), 19(R)-hydroxy prostaglandin E, (Cayman
Chemical, Ann Arbor, MI, USA) and indomethacin (Dumex, Copenhagen, Denmark) were used.
RESULTS Nerve stimulation Hypogastric nerve stimulation increased secre10; n = 7) (Figs 1, 2 & 3). tion by 113% (i-
Drug efects on hypogastric nerve stirnulation induced secretion (Figs 1 6 2) Prostaglandin I, ( 10P7-10-' M) as well as prostaglandin E, ( 10-7-10-5 M) prominently inhibited nerve stimulation induced secretion (P < 0.05). Prostaglandin F,, ( 10-7-10-5 M) also markedly depressed nerve induced secretion (P< 0.05), and 19-OH PGE, (loP6M) reduced secretory responses to a level of 31 yo(k6 ; n = 6) ( P < 0.05 vs. controls and other prostaglandins). Phentolamine (loP6 M) did not reverse the inhibitory effect of PGF,, (lo-' M) on hypo-
+
5-2
132
B.Jonsson and M . Hummarstrom 7
J Fig. 3. Effects of prostaglandins on resting secretion level from cervical glands at ovulation time. Hypogastric nerve stimulation [controls increased secretion by 113?b. Prostaglandin I, (m 10." M) enhanced secretion to the same extent as did nerve stimulation (n.s. vs. controls, P < 0.05 vs. PGE, and PGFJ. Also 19-OH PGE, ( @ M) had a marked stimulatory effect on secretory linings (P < 0.05 s's. controls, PGE, and PGF,J. Prostaglandin E, (@ 10-7-10-5 M) as well as PGF,, (g 10-'-10-' M) did not induce an>- carbohydrate secretion. Administration of the adrenergic blocker phentolamine X)I in combination with PGF,, (m) resulted in a marked secretory response ( P < 0.0-5 s's. PGF,z; 10-'-10-" 51). (Mean and SER.1; number of experiments indicated by figures a t columns.)
(o)]
gastric nerve stimulation ( - 1 0 1 4 6 ; IZ = 4) (Fig. 1). Indomethacin (10-'-10-' hi) (Fig. 2 ) dose-dependent11 inhibited the nerve stimulation induced secretion ( P < 0.05).
Drug ejects on basal secretion (Figs2 6 3) Prostaglandin I, resulted in a dose dependent secretory response, and lo-' bi PGI, increased secretion to the same extent as did nerve stimulation ( 76", i 18; n = 4). 19-OH PGE, (10- li M) also resulted in an increase of secretion (+.5joo& 18; n = 6 ; P < 0.05 vs. controls and PGE:,, PGF,,). Prostaglandin E, ( 10-7-10-~5\I) and PGF,, (10-'-10-' M) had no effects on cervical secretion ( P < 0.05). T h e combination M) and PGF,, ( M) of phentolamine ( resulted in a secretory response of 4000 ( f 18; ti = 6 ; P < 0.05 vs. controls and PGF,, M) (Fig. 3 ) . Indomethacin (10-7-10-' M) did not y-ield any carbohydrate secretion (Fig. 2 ) .
+
+
DISCUSSION Prostaglandins, known potent neuromodulators of autonomic nervous transmission, are synthesized in and exert different and contradictory effects on the uterus. I n the human a high concentration of prostaglandins are present as a result of the seminal fluid deposed close to the uterus. Semen of several species as e.g. the guinea-pig is considered to contain low levels of prostaglandins (Eliasson 1959, Cenedella 1975). Even small amounts of prostaglandins are, however, supposed to exert biological effects (Cenedella 1975). Despite methodological obstacles it was supposed to be of interest to study the effects of prostaglandins and a prostaglandin H synthase inhibitor on cervical secretion, which is supposed to be of great importance for fertilization. An enhancement of cholinergic neurotransmission after application of prostaglandins has
Prostaglandins injuence cervix secretion been shown (Gustafsson 1989). Furthermore, PGF,, has been shown to exert secretory effects on salivary glands (Hahn & Patil 1972). In this study, application of 19-OH PGE, resulted in an apparent secretory response. Also PGI, induced a secretory response. The effect is probably postjunctional as nerve stimulation in combination with these prostaglandins does not result in any prominent secretory effect. Furthermore, prejunctional stimulation of cholinergic neurons is rarely demonstrated (Gustafsson 1989). Also Hahn & Patil (1974) suggest a postjunctional secretory effect by PGF,, since tetrodotoxin does not reduce the secretory response induced in salivary glands. Prostaglandin E, does not have any secretory effects and this is in line with findings of PGE, on gastric secretion (Sautereau et al. 1989). All prostaglandins except 19-OH PGE, abolished nerve stimulation induced secretion. The inhibitory effect of prostaglandins on nerve stimulation induced secretion might represent stimulation of adrenergic nerves thus unmasking an adrenergic inhibition. In the endometrium of the guinea-pig an a-adrenergic inhibitory innervation has been shown although no adrenergic influence on nerve stimulation induced secretion from the cervical glands has been demonstrated (Hammarstrom 1985, 1989). The adrenergic inhibition of cholinergic secretomotor innervation of the endometrium was proven after postganglionic nerve stimulation and in studies on cervical secretion (Hammarstrom 1989) nerve stimulation was performed on isolated nerve preparations. Lack of demonstrable adrenergic inhibition in this hypogastric nerve-cervix uteri preparation might be due to the fact that nerve stimulation was mainly performed preganglionic and thus resulted in divergence in the synaptic region. The adrenergic inhibition might therefore be difficult to demonstrate. Concomitantly, the secretory responses to PGI, and PGF,, could be due to inhibition of adrenergic transmission, since secretory response to PGF,, is much enhanced after administration of the a-adrenergic inhibitor phentolamine. Prostaglandin I, and F,, primarily have adrenergic stimulatory effects (Gustafsson 1989). Indomethacin did not exert any secretory effects on cervical glands and further dosedependently inhibited nerve induced secretion. Indomethacin has been discussed as a potent calcium antagonist and it is well settled that
133
indomethacin effects could not to a full extent be reversed by prostaglandin application (Gustafsson 1989). The many pitfalls in studying prostaglandin modulation effects on autonomic neurotransmission is extensively reviewed by Gustafsson (1989). In this preparation there is also the complex peripheral autonomic nervous supply to the paracervical ganglia and uterus described in the review of Papka et al. (1987). Furthermore, metabolism of prostaglandins in this in vitro preparation could not be excluded (Christensen 1984). In conclusion, PGI, and 19-OH PGE, exert effects on basal secretion from cervical glands and seem to be potent postjunctional stimulators. Marked reduction of nerve stimulation induced secretion is seen after all prostaglandins, except after 19-OH PGE, which to a lesser extent reduced secretory responses. Indomethacin, frequently used in pharmacological doses as pain relief among fertile women, is a potent inhibitor of secretion induced by nerve stimulation. Thus, in this in vitro study metabolites of the arachidonic acid cascade and its inhibitor can modulate cervical secretion and thus maybe influence fertilization. The present study was supported by grants from the Karolinska Institute and the Swedish Medical Society. For excellent technical assistance we thank Ms Astrid
Haggblad.
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