Acta Physiol. Scand. 1975. 95. 142-144 From the Department of Physiology, Karolinska Institutet, Stockholm, Sweden
Noradrenaline Release Following Nerve Stimulation and its Modification by Prostaglandin E2in Human and Rabbit Oviduct BY
PERHEDQVIST and MARCBYGDEMAN ATEFMOAWAD, The oviduct muscular wall at the isthmus and especially at the ampullary isthmic junction (AIJ) is heavily innervated with adrenergic fibres in all mammalian species studied, including man (Brundin 1965, Owman et al. 1967). The importance of the neuromuscular mechanisms in this region in the physiology of ovum transport has been the subject of many recent studies. Locking of the fertilized ovum at the AIJ for 48-72 h is said to be governed by the adrenergic control mechanisms (Pauerstein 1974). Moreover prostaglandins (PGs) have been implicated in the modification of the physiological phenomena of this organ (Spillman 1974). The effects of PGs on the release of noradrenaline (NA) have been extensively studied in other organs (cf. Hedqvist 1974, Frame and Hedqvist 1975). The subject of this report is the study of the effects of nerve stimulation on the neurotransmitter release, its modification by prostaglandins and the parallel effector responses in oviductal isthmus at the AIJ in the rabbit and the human. The isthmic part of human and rabbit oviducts, l&-2 cm in length, was preincubated for one hour in Tyrode’s solution containing 2.5 ,uCi/ml3H-(-)-NA(sp.a. 5 Ci/mmol,N.E.N.). It was then mounted in a 2 ml bath in a longitudinal orientation and was superfused with Tyrode at a rate of 1.5 ml/min. The composition of the Tyrode was (conc. in mM): NaCl 136.7, KC1 2.7, CaCI, 1.8, MgCI, 1.0, NaHCO, 11.9, NaHPO, 0.4, glucose 5.5, ascorbic acid 0.1, and it was kept at 37°C and was gassed with 5 % CO, in 0,. The organ was transmurally stimulated by means of platinum wire electrodes in the wall of the bath and a Grass S4 stimulator delivering trains of biphasic pulses (5 Hz, 1 ms, supramaximal voltage) at 10-15 min intervals. Contractions were recorded by an isotonic transducer (Harvard 354), loaded with 0.5-1.0 g, and a Grass Model 5 polygraph. The superfusate was divided into one minute samples and the radioactivity was determined by counting 1 rnl aliquots in a Packard liquid scintillation spectrometer using 10 ml Instagel as counting medium. Quenching was monitored by internal standards. Prostaglandins were kindly supplied by Dr John Pike, Upjohn Co, Kalarnazoo, U.S.A. Transmural stimulation of superfused human and rabbit oviducts for 30-60 s periods (5 Hz, 1 ms, supramaximal voltage) consistently caused a marked and well reproducible increase in the release of tracer, the bulk of which consisted of intact 3H-NA. The stimulation parameters, as well as the finding that tetrodotoxin (0.2 pg/ml) and guanethidine (2 ,ug/ml) prevented the stimulation induced release of tracer and the resultant effector response, indicate activation of adrenergic nerve fibres. Independent of hormonal condition, pregnancy, estrus or progesterone dominance, the effector response to transmural nerve stimulation in the rabbit oviduct was consistently that of contraction. In the human oviduct, however, the effector response was variable, i.e. a contraction, no response or even marked relaxation. Presumably, and in contrast to the 142
143
PGEi O N NORADRENALlNE RELEASE IN OVlDUCT
1500
0 r 0
5 1250 2
I L
.$ 1000 u. x 3
Y J
w I
Fig. 1. Human oviduct previously loaded with *H-NA. Effect of PGE, on the outflow of tracer in response to transmural stimulation (NS), 300 pulses at 5 Hz. Time in minutes =fraction numbers.
?SO
'r
'0° 0
10 NS
20 NS
30
t
40
50
NS
60
t
t
NS
NS
70
80
FRACTION NUMBER
rabbit, this is related to the hormonal condition of the subject, since it has already been shown that estrogen predominance enhances a-adrenergic receptor activity while progesterone that of the &receptors (Moawad and Kim 1974). In all hormonal conditions in the rabbit as well as in the human, PGE, in doses ranging from 9 x 10-e M to 9 x M inhibited the release of 3H-NA in response to transmural nerve stimulation. The degree of inhibition was dose-dependent and the effect clearly reversible (Fig. 1). In the rabbit oviduct of both estrogen and progesterone dominated animals PGE, caused inhibition of spontaneous motility. The contractions resulting from transmural nerve stimulation were also consistently reduced. The depression of both the spontaneous activity and the nerve stimulation responses were dose-dependent as well (Fig. 2). In the human, in spite of the consistent inhibition of $H-NA release, PGE, modified the oviductal motility in various ways. While spontaneous motility, when present, always was markedly depressed or abolished, contractions resulting from nerve stimulation were either slightly depressed, not affected o r even markedly enhanced. The one experiment where this latter effect is well demonstrated is shown in Fig. 3. This particular tissue was obtained from a subject in the immediate pre-ovulatory period. Presumably, therefore, her estrogen production was at a good level in conjunction with insignificant progesterone levels. When the effector response to transmural nerve stimulation was that of depression, the possible influence by PGEl should not be settled because of the almost complete cessation of 10 rnin t (
PGE2 Fig. 2. Effect of PGE, on the isotonic contractions of a rabbit oviduct. Transmural nerve stimulation (NS) 50 pulses at 5 Hz were delivered at 5 min intervals.
PGE2
J.7iZFK.l I
NS
.~
JxGiGl L
i 1 . I
F,
f f f f ? ? ? f ? f ? f' ?' f ? f f
144
ATEF MOAWAD, PER HEDQVIST AND MARC BYGDEMAN
n 9
NS
t
NS
t
NS
t
NS
t
NS
5 min
f
NS
T
NS
T
NS
Fig. 3. Effect of PGE, on the isotonic contractions of a human oviduct in the late follicular phase. Transmural stimulation (NS) 300 pulses at 5 Hz were delivered at 5 min intervals.
spontaneous activity in the presence of PGEa. However, in n o case, was the relaxation by nerve stimulation converted into one of contraction.
Comment The isthmic region of human and rabbit oviduct is heavily innervated with adrenergic neurons. We have shown in these experiments that nerve stimulation results in increased N A release in both species. The invariable inhibition of this NA release by the addition of small doses of PGE, is similar t o results obtained from other adrenergically innervated tissues (cf. Hedqvist 1974, Frame and Hedqvist 1975). In the rabbit oviduct there was a parallelism between restriction of transmitter release and inhibition of effector response. The finding that nerve stimulation induced contractions of the human oviduct sometimes were enhanced by PGE1, in spite of depressed transmitter release, is unexpected. I t points out the importance of species differences. Also, the resultant response in the human oviduct might well be related t o the hormonal background of the subject. Further detailed studies correlating the various responses t o the hormone levels are therefore essential. This study was supported by grants from the Swedish Medical Research Council, project 04X-4243. A.M. is on sabbatical leave from University of Chicago, supported by the Rockefeller Foundation.
References BRUNDIN, J., Distribution and function of adrenergic nerves in the rabbit Fallopian tube. Acta physiol. scand. 1965. 66. Suppl. 259. 1-57. FRAME, M. H. and P. HEDQVIST, Evidence for prostaglandin mediated prejunctional control of renal vascular sympathetic tone. Brit. J. Pharmacol. 1975. 54. 189-196. HEDQVIST, P., Effect of prostaglandins and prostaglandin synthesis inhibitors on norepinephrine release from vascular tissue. In Prostaglandin Synrhetase Inhibitors, eds. Robinson, H. J. and Vane, J. R., pp. 303-309. Raven Press, New York. 1974. MOAWAD, A. H. and M. KIM,Plasma sex steroid levels and the in uirro response of the isthmic portion of human oviduct to catecholamines. Cynec. Invest. 1974. 5. 19-27. and N. 0. SJOBERG, Adrenergic innervation of the human female OWMAN,CH., E. ROSENCREN reproductive organs: a histochemical and chemical investigation. Obstet. Cynecol. 1967. 30. 763773. PAUERSTEIN, C . J., T i e Fallopian Tube: A Reappraisal. Lea and Febiger, Philadelphia, 1974. SPILLMAN, C. H., Oviduct response to prostaglandins: influence of estradiol and progesterone. Prostaglandins 1974. 7. 465469.
Noradrenaline Release Following Nerve Stimulation and its Modification by Prostaglandin E2in Human and Rabbit Oviduct BY
PERHEDQVIST and MARCBYGDEMAN ATEFMOAWAD, The oviduct muscular wall at the isthmus and especially at the ampullary isthmic junction (AIJ) is heavily innervated with adrenergic fibres in all mammalian species studied, including man (Brundin 1965, Owman et al. 1967). The importance of the neuromuscular mechanisms in this region in the physiology of ovum transport has been the subject of many recent studies. Locking of the fertilized ovum at the AIJ for 48-72 h is said to be governed by the adrenergic control mechanisms (Pauerstein 1974). Moreover prostaglandins (PGs) have been implicated in the modification of the physiological phenomena of this organ (Spillman 1974). The effects of PGs on the release of noradrenaline (NA) have been extensively studied in other organs (cf. Hedqvist 1974, Frame and Hedqvist 1975). The subject of this report is the study of the effects of nerve stimulation on the neurotransmitter release, its modification by prostaglandins and the parallel effector responses in oviductal isthmus at the AIJ in the rabbit and the human. The isthmic part of human and rabbit oviducts, l&-2 cm in length, was preincubated for one hour in Tyrode’s solution containing 2.5 ,uCi/ml3H-(-)-NA(sp.a. 5 Ci/mmol,N.E.N.). It was then mounted in a 2 ml bath in a longitudinal orientation and was superfused with Tyrode at a rate of 1.5 ml/min. The composition of the Tyrode was (conc. in mM): NaCl 136.7, KC1 2.7, CaCI, 1.8, MgCI, 1.0, NaHCO, 11.9, NaHPO, 0.4, glucose 5.5, ascorbic acid 0.1, and it was kept at 37°C and was gassed with 5 % CO, in 0,. The organ was transmurally stimulated by means of platinum wire electrodes in the wall of the bath and a Grass S4 stimulator delivering trains of biphasic pulses (5 Hz, 1 ms, supramaximal voltage) at 10-15 min intervals. Contractions were recorded by an isotonic transducer (Harvard 354), loaded with 0.5-1.0 g, and a Grass Model 5 polygraph. The superfusate was divided into one minute samples and the radioactivity was determined by counting 1 rnl aliquots in a Packard liquid scintillation spectrometer using 10 ml Instagel as counting medium. Quenching was monitored by internal standards. Prostaglandins were kindly supplied by Dr John Pike, Upjohn Co, Kalarnazoo, U.S.A. Transmural stimulation of superfused human and rabbit oviducts for 30-60 s periods (5 Hz, 1 ms, supramaximal voltage) consistently caused a marked and well reproducible increase in the release of tracer, the bulk of which consisted of intact 3H-NA. The stimulation parameters, as well as the finding that tetrodotoxin (0.2 pg/ml) and guanethidine (2 ,ug/ml) prevented the stimulation induced release of tracer and the resultant effector response, indicate activation of adrenergic nerve fibres. Independent of hormonal condition, pregnancy, estrus or progesterone dominance, the effector response to transmural nerve stimulation in the rabbit oviduct was consistently that of contraction. In the human oviduct, however, the effector response was variable, i.e. a contraction, no response or even marked relaxation. Presumably, and in contrast to the 142
143
PGEi O N NORADRENALlNE RELEASE IN OVlDUCT
1500
0 r 0
5 1250 2
I L
.$ 1000 u. x 3
Y J
w I
Fig. 1. Human oviduct previously loaded with *H-NA. Effect of PGE, on the outflow of tracer in response to transmural stimulation (NS), 300 pulses at 5 Hz. Time in minutes =fraction numbers.
?SO
'r
'0° 0
10 NS
20 NS
30
t
40
50
NS
60
t
t
NS
NS
70
80
FRACTION NUMBER
rabbit, this is related to the hormonal condition of the subject, since it has already been shown that estrogen predominance enhances a-adrenergic receptor activity while progesterone that of the &receptors (Moawad and Kim 1974). In all hormonal conditions in the rabbit as well as in the human, PGE, in doses ranging from 9 x 10-e M to 9 x M inhibited the release of 3H-NA in response to transmural nerve stimulation. The degree of inhibition was dose-dependent and the effect clearly reversible (Fig. 1). In the rabbit oviduct of both estrogen and progesterone dominated animals PGE, caused inhibition of spontaneous motility. The contractions resulting from transmural nerve stimulation were also consistently reduced. The depression of both the spontaneous activity and the nerve stimulation responses were dose-dependent as well (Fig. 2). In the human, in spite of the consistent inhibition of $H-NA release, PGE, modified the oviductal motility in various ways. While spontaneous motility, when present, always was markedly depressed or abolished, contractions resulting from nerve stimulation were either slightly depressed, not affected o r even markedly enhanced. The one experiment where this latter effect is well demonstrated is shown in Fig. 3. This particular tissue was obtained from a subject in the immediate pre-ovulatory period. Presumably, therefore, her estrogen production was at a good level in conjunction with insignificant progesterone levels. When the effector response to transmural nerve stimulation was that of depression, the possible influence by PGEl should not be settled because of the almost complete cessation of 10 rnin t (
PGE2 Fig. 2. Effect of PGE, on the isotonic contractions of a rabbit oviduct. Transmural nerve stimulation (NS) 50 pulses at 5 Hz were delivered at 5 min intervals.
PGE2
J.7iZFK.l I
NS
.~
JxGiGl L
i 1 . I
F,
f f f f ? ? ? f ? f ? f' ?' f ? f f
144
ATEF MOAWAD, PER HEDQVIST AND MARC BYGDEMAN
n 9
NS
t
NS
t
NS
t
NS
t
NS
5 min
f
NS
T
NS
T
NS
Fig. 3. Effect of PGE, on the isotonic contractions of a human oviduct in the late follicular phase. Transmural stimulation (NS) 300 pulses at 5 Hz were delivered at 5 min intervals.
spontaneous activity in the presence of PGEa. However, in n o case, was the relaxation by nerve stimulation converted into one of contraction.
Comment The isthmic region of human and rabbit oviduct is heavily innervated with adrenergic neurons. We have shown in these experiments that nerve stimulation results in increased N A release in both species. The invariable inhibition of this NA release by the addition of small doses of PGE, is similar t o results obtained from other adrenergically innervated tissues (cf. Hedqvist 1974, Frame and Hedqvist 1975). In the rabbit oviduct there was a parallelism between restriction of transmitter release and inhibition of effector response. The finding that nerve stimulation induced contractions of the human oviduct sometimes were enhanced by PGE1, in spite of depressed transmitter release, is unexpected. I t points out the importance of species differences. Also, the resultant response in the human oviduct might well be related t o the hormonal background of the subject. Further detailed studies correlating the various responses t o the hormone levels are therefore essential. This study was supported by grants from the Swedish Medical Research Council, project 04X-4243. A.M. is on sabbatical leave from University of Chicago, supported by the Rockefeller Foundation.
References BRUNDIN, J., Distribution and function of adrenergic nerves in the rabbit Fallopian tube. Acta physiol. scand. 1965. 66. Suppl. 259. 1-57. FRAME, M. H. and P. HEDQVIST, Evidence for prostaglandin mediated prejunctional control of renal vascular sympathetic tone. Brit. J. Pharmacol. 1975. 54. 189-196. HEDQVIST, P., Effect of prostaglandins and prostaglandin synthesis inhibitors on norepinephrine release from vascular tissue. In Prostaglandin Synrhetase Inhibitors, eds. Robinson, H. J. and Vane, J. R., pp. 303-309. Raven Press, New York. 1974. MOAWAD, A. H. and M. KIM,Plasma sex steroid levels and the in uirro response of the isthmic portion of human oviduct to catecholamines. Cynec. Invest. 1974. 5. 19-27. and N. 0. SJOBERG, Adrenergic innervation of the human female OWMAN,CH., E. ROSENCREN reproductive organs: a histochemical and chemical investigation. Obstet. Cynecol. 1967. 30. 763773. PAUERSTEIN, C . J., T i e Fallopian Tube: A Reappraisal. Lea and Febiger, Philadelphia, 1974. SPILLMAN, C. H., Oviduct response to prostaglandins: influence of estradiol and progesterone. Prostaglandins 1974. 7. 465469.
