Br. J. Pharmacol. (1991), 103, 1429-1434
(D Macmillan Press Ltd, 1991
Response of the rat myometrium to phenylephrine in early pregnancy and the effects of 6-hydroxydopamine Andrew Kaulenas, 'Helena C. Parkington & Harold A. Coleman Department of Physiology, Monash University, Clayton, Victoria, 3168, Australia 1 The contractile responses of the longitudinal and circular muscle layers of the rat uterus to the ax-adrenoceptor agonist phenylephrine were measured on days 3-6 of gestation. There was a progressive increase in sensitivity to phenylephrine in both muscle layers between days 3 and 6 of gestation. Overall, this amounted to a 13 and 9 fold increase in sensitivity in longitudinal and circular muscles, respectively. In longitudinal muscle the slope of the Hill plot was 2 on day 3 of pregnancy and was decreased to 1 thereafter. 2 The sympathetic nerve terminals innervating the smooth muscle of the uterus were destroyed by administration of 6-hydroxydopamine (2 x 50mgkg-1) 4-7 days before testing with phenylephrine. Following this treatment there was a significant increase in sensitivity to phenylephrine on day 3 in both muscle layers. After day 4, the longitudinal muscle was less sensitive to phenylephrine. 3 In the longitudinal muscle there was a progressive increase in the contractile response to maximal concentrations of phenylephrine and to high potassium (100mM) between days 3 and 6 of pregnancy. In the circular muscle the responsiveness to both phenylephrine and potassium remained unchanged between days 3 and 6 of gestation. 6-Hydroxydopamine had no effect on the maximal responses to phenylephrine or high potassium in either muscle layer. 4 In conclusion, denervation supersensitivity of uterine smooth muscle following injection of 6hydroxydopamine is observed only on day 3 of pregnancy and appears to be replaced by subsensitivity by day 6. The decrease in the slope of the Hill plot in longitudinal muscle after day 3 may be explained by changes in events between activation of a1-adrenoceptors and contraction. Keywords: Denervation supersensitivity; myometrium; drug effects; blastocyst spacing; sympathectomy, chemical; hydroxydopamines; uterus, rat; sympathomimetics; blastocyst, peri-implantation phase
Introduction It is likely that the contractile activity of uterine smooth muscle plays an important role in the spacing of blastocysts along the uterus in polytocous species (Boving, 1956; McLaren & Michie, 1959; Pusey et al., 1980; Legrand et al., 1987). The influence of the sympathetic innervation on the contractile activity of uterine smooth muscle around the time of implantation has been studied in rats (Roche et al., 1985; Legrand et al., 1987; 1989). Surgical destruction of the ovarian nerve had no effect on either blastocyst transport or spacing (Roche et al., 1985). Injection of the a-adrenoceptor antagonists, prazosin and phenoxybenzamine, depressed electromyographical activity in the uterus of rats (Legrand et al., 1989), disrupted transport and implantation, and even spacing of foetuses did not occur (Legrand et al., 1987). The available evidence suggests that the cholinergic nerves provide most of the motor innervation of the uterus (Hollingsworth, 1974; Sato et al., 1989). Prazosin interacts with the presynaptic control of some cholinergic nerves (Koss et al., 1990; Thompson et al., 1990) and this could explain the actions of this drug on the uterus. Alternatively, since blastocyst transport and spacing remained unaffected following injection of 6hydroxydopamine to ablate the noradrenergic nerves in the reproductive tract of rats, the development of supersensitivity in the muscle of the uterus was proposed (Legrand et al., 1987). It was postulated that, in such an event, low levels of catecholamines in the circulation might be sufficient to maintain normal uterine motility during this critical period of blastocyst transport, spacing and implantation (Legrand et al., 1987). In the present study, we set out to determine whether supersensitivity in response to a-adrenoceptor stimulation followed denervation with 6-hydroxydopamine in rat uterine smooth muscle in the peri-implantation period. The two muscle layers I
Author for correspondence.
of the uterus were studied separately because they do not behave in the same way, even under identical hormonal conditions (Osa & Katase, 1975) and the population of adrenoceptors varies between the two layers (Kawarabayashi & Osa, 1976).
Methods Sprague-Dawley rats were divided randomly into two groups: Group I Pairs of virgin females were housed with one male and vaginal washings from the females were examined daily. The presence of spermatozoa in the vaginal smear was denoted as day I of pregnancy. Uterine activity was studied on one of days 3 to 6 of pregnancy since the blastocysts arrive in the uterus around mid-day on day 4 and become implanted within 24 h (Forcelledo et al., 1981; Legrand et al., 1987). These rats served as controls.
Group 2 Females were injected intraperitoneally with 6hydroxydopamine 50mgkg-1 on two consecutive days and then mated as for group 1. Uterine activity was examined 4-7 days following the last injection to ensure that maximum supersensitivity had become established (Furness et al., 1970). The females were killed by decapitation. A segment from each uterine horn was removed immediately and frozen at - 20'C for visualization of noradrenaline-containing nerve varicosities using fluorescence histochemistry. Strips (7-12mm long and 1.5-2mm wide) of longitudinal and circular muscle were prepared. A truly circular strip of muscle is generally 2-4mm in length at this stage of pregnancy, so a tight spiral strip was cut in order to obtain the
A. KAULENAS et al.
1430
greater length. A silk thread was tied to each end of each strip. The two strips of tissue from each rat were mounted in tissue chambers. One end of each was fixed while the other end was connected to an isometric force transducer (FT03, Grass). The chambers were continuously perfused at 3 ml minm ' with oxygenated physiological solution containing (mM): NaCl 120, KCI 5, CaCl2 2.5, MgSO4 1, KH2PO4 1.2, NaHCO3 25 and glucose 11, gassed with 95% 02, 5% CO2 and maintained at 370C. Both strips were stretched to approximately 0.2 g tension since this resulted in the production of >80% of maximal contraction in response to solution containing 100mm potassium. Isometric tension was recorded on a chart recorder (Grass) and passed to a computer which digitized the signal for subsequent integration of the responses. An equilibration period of about 1 h was allowed in order to stretch the preparations and for spontaneous activity to settle down to a constant level. For most experiments propranolol (10-6M) was added to the perfusate during the final 30 min of the equilibration period and for the remainder of the experiment in order to block f-adrenoceptors. Stimulation of a-adrenoceptors was achieved by perfusing the tissues with phenylephrine (10'8 to 10-4M applied in random order) for 5 min with 15-20 min allowed for recovery before the next dose was administered. It was determined in pilot experiments that desensitization to phenylephrine did not occur under this regime. The response of the myometrium to phenylephrine is complex, particularly in the circular layer, with variations in the amplitude, frequency and duration of the phasic contractions. The response was assessed by integrating the area under the contraction curve which is a function of these three parameters. This was achieved by use of the computer software package pClamp (Axon Instruments). The integral of the spontaneous contractility during the 5min immediately preceding was subtracted from the integral of the response to phenylephrine (Figure la). The result was normalized as a % of the maximum response to phenylephrine, gena
erally 10- M. A dose-response curve was constructed from the data for each individual animal i.e. a sigmoid curve was fitted to the data with the computer software package, GraphPad (ISI Software) and an EC50 was calculated (Figure lb). Data from 5-6 animals were examined for each day of pregnancy (days 3-6), and for each treatment, from which a mean EC50 was obtained. The same software was used to calculate the slope of the Hill plot for each set of data. In order to determine the capacity of the strips to contract in absolute terms, the cross-sectional area of each strip was calculated from the length and mass of muscle between the threads, measured at the end of each experiment. The contractile response to the maximal phenylephrine concentration was normalized per unit cross-sectional area of the muscle strips. It has been observed that supersensitivity in many smooth muscle tissues may also extend to stimulatory agents other than the specific agonists under consideration, for example, to potassium (Westfall, 1981). Thus, the contractile responses to solutions containing 100 mm potassium (KCl replaced the isosmotic equivalent of NaCl in physiological solution) were also investigated in the present study. Transverse sections (15,pm thick and 60-100 from each animal) of the frozen segments of uterus were stained with glyoxylic acid (de la Torre & Surgeon, 1976) and viewed under a fluorescence microscope to visualize noradrenalinecontaining nerves. Hence, the presence of nerves in intact tissues or the effectiveness of denervation following injection of 6-hydroxydopamine was established for each preparation. Data from different groups were tested by analyses of variance (ANOVA). The effects of 6-hydroxydopamine were analyzed by comparing EC50 values to phenylephrine. Similar tests were used to determine the significance of responsiveness to maximal concentrations of phenylephrine and 100 mm potassium. The mean and standard error is given and a significance level of 0.05 was used in testing. (-)-Phenylephrine hydrochloride, 6-hydroxydopamine hydrobromide and (± )-propranolol hydrochloride were obtained from Sigma Chemical Company, U.S.A.
1500 g sec 0 I4 g
10.2 g
\I
/
(D Macmillan Press Ltd, 1991
Response of the rat myometrium to phenylephrine in early pregnancy and the effects of 6-hydroxydopamine Andrew Kaulenas, 'Helena C. Parkington & Harold A. Coleman Department of Physiology, Monash University, Clayton, Victoria, 3168, Australia 1 The contractile responses of the longitudinal and circular muscle layers of the rat uterus to the ax-adrenoceptor agonist phenylephrine were measured on days 3-6 of gestation. There was a progressive increase in sensitivity to phenylephrine in both muscle layers between days 3 and 6 of gestation. Overall, this amounted to a 13 and 9 fold increase in sensitivity in longitudinal and circular muscles, respectively. In longitudinal muscle the slope of the Hill plot was 2 on day 3 of pregnancy and was decreased to 1 thereafter. 2 The sympathetic nerve terminals innervating the smooth muscle of the uterus were destroyed by administration of 6-hydroxydopamine (2 x 50mgkg-1) 4-7 days before testing with phenylephrine. Following this treatment there was a significant increase in sensitivity to phenylephrine on day 3 in both muscle layers. After day 4, the longitudinal muscle was less sensitive to phenylephrine. 3 In the longitudinal muscle there was a progressive increase in the contractile response to maximal concentrations of phenylephrine and to high potassium (100mM) between days 3 and 6 of pregnancy. In the circular muscle the responsiveness to both phenylephrine and potassium remained unchanged between days 3 and 6 of gestation. 6-Hydroxydopamine had no effect on the maximal responses to phenylephrine or high potassium in either muscle layer. 4 In conclusion, denervation supersensitivity of uterine smooth muscle following injection of 6hydroxydopamine is observed only on day 3 of pregnancy and appears to be replaced by subsensitivity by day 6. The decrease in the slope of the Hill plot in longitudinal muscle after day 3 may be explained by changes in events between activation of a1-adrenoceptors and contraction. Keywords: Denervation supersensitivity; myometrium; drug effects; blastocyst spacing; sympathectomy, chemical; hydroxydopamines; uterus, rat; sympathomimetics; blastocyst, peri-implantation phase
Introduction It is likely that the contractile activity of uterine smooth muscle plays an important role in the spacing of blastocysts along the uterus in polytocous species (Boving, 1956; McLaren & Michie, 1959; Pusey et al., 1980; Legrand et al., 1987). The influence of the sympathetic innervation on the contractile activity of uterine smooth muscle around the time of implantation has been studied in rats (Roche et al., 1985; Legrand et al., 1987; 1989). Surgical destruction of the ovarian nerve had no effect on either blastocyst transport or spacing (Roche et al., 1985). Injection of the a-adrenoceptor antagonists, prazosin and phenoxybenzamine, depressed electromyographical activity in the uterus of rats (Legrand et al., 1989), disrupted transport and implantation, and even spacing of foetuses did not occur (Legrand et al., 1987). The available evidence suggests that the cholinergic nerves provide most of the motor innervation of the uterus (Hollingsworth, 1974; Sato et al., 1989). Prazosin interacts with the presynaptic control of some cholinergic nerves (Koss et al., 1990; Thompson et al., 1990) and this could explain the actions of this drug on the uterus. Alternatively, since blastocyst transport and spacing remained unaffected following injection of 6hydroxydopamine to ablate the noradrenergic nerves in the reproductive tract of rats, the development of supersensitivity in the muscle of the uterus was proposed (Legrand et al., 1987). It was postulated that, in such an event, low levels of catecholamines in the circulation might be sufficient to maintain normal uterine motility during this critical period of blastocyst transport, spacing and implantation (Legrand et al., 1987). In the present study, we set out to determine whether supersensitivity in response to a-adrenoceptor stimulation followed denervation with 6-hydroxydopamine in rat uterine smooth muscle in the peri-implantation period. The two muscle layers I
Author for correspondence.
of the uterus were studied separately because they do not behave in the same way, even under identical hormonal conditions (Osa & Katase, 1975) and the population of adrenoceptors varies between the two layers (Kawarabayashi & Osa, 1976).
Methods Sprague-Dawley rats were divided randomly into two groups: Group I Pairs of virgin females were housed with one male and vaginal washings from the females were examined daily. The presence of spermatozoa in the vaginal smear was denoted as day I of pregnancy. Uterine activity was studied on one of days 3 to 6 of pregnancy since the blastocysts arrive in the uterus around mid-day on day 4 and become implanted within 24 h (Forcelledo et al., 1981; Legrand et al., 1987). These rats served as controls.
Group 2 Females were injected intraperitoneally with 6hydroxydopamine 50mgkg-1 on two consecutive days and then mated as for group 1. Uterine activity was examined 4-7 days following the last injection to ensure that maximum supersensitivity had become established (Furness et al., 1970). The females were killed by decapitation. A segment from each uterine horn was removed immediately and frozen at - 20'C for visualization of noradrenaline-containing nerve varicosities using fluorescence histochemistry. Strips (7-12mm long and 1.5-2mm wide) of longitudinal and circular muscle were prepared. A truly circular strip of muscle is generally 2-4mm in length at this stage of pregnancy, so a tight spiral strip was cut in order to obtain the
A. KAULENAS et al.
1430
greater length. A silk thread was tied to each end of each strip. The two strips of tissue from each rat were mounted in tissue chambers. One end of each was fixed while the other end was connected to an isometric force transducer (FT03, Grass). The chambers were continuously perfused at 3 ml minm ' with oxygenated physiological solution containing (mM): NaCl 120, KCI 5, CaCl2 2.5, MgSO4 1, KH2PO4 1.2, NaHCO3 25 and glucose 11, gassed with 95% 02, 5% CO2 and maintained at 370C. Both strips were stretched to approximately 0.2 g tension since this resulted in the production of >80% of maximal contraction in response to solution containing 100mm potassium. Isometric tension was recorded on a chart recorder (Grass) and passed to a computer which digitized the signal for subsequent integration of the responses. An equilibration period of about 1 h was allowed in order to stretch the preparations and for spontaneous activity to settle down to a constant level. For most experiments propranolol (10-6M) was added to the perfusate during the final 30 min of the equilibration period and for the remainder of the experiment in order to block f-adrenoceptors. Stimulation of a-adrenoceptors was achieved by perfusing the tissues with phenylephrine (10'8 to 10-4M applied in random order) for 5 min with 15-20 min allowed for recovery before the next dose was administered. It was determined in pilot experiments that desensitization to phenylephrine did not occur under this regime. The response of the myometrium to phenylephrine is complex, particularly in the circular layer, with variations in the amplitude, frequency and duration of the phasic contractions. The response was assessed by integrating the area under the contraction curve which is a function of these three parameters. This was achieved by use of the computer software package pClamp (Axon Instruments). The integral of the spontaneous contractility during the 5min immediately preceding was subtracted from the integral of the response to phenylephrine (Figure la). The result was normalized as a % of the maximum response to phenylephrine, gena
erally 10- M. A dose-response curve was constructed from the data for each individual animal i.e. a sigmoid curve was fitted to the data with the computer software package, GraphPad (ISI Software) and an EC50 was calculated (Figure lb). Data from 5-6 animals were examined for each day of pregnancy (days 3-6), and for each treatment, from which a mean EC50 was obtained. The same software was used to calculate the slope of the Hill plot for each set of data. In order to determine the capacity of the strips to contract in absolute terms, the cross-sectional area of each strip was calculated from the length and mass of muscle between the threads, measured at the end of each experiment. The contractile response to the maximal phenylephrine concentration was normalized per unit cross-sectional area of the muscle strips. It has been observed that supersensitivity in many smooth muscle tissues may also extend to stimulatory agents other than the specific agonists under consideration, for example, to potassium (Westfall, 1981). Thus, the contractile responses to solutions containing 100 mm potassium (KCl replaced the isosmotic equivalent of NaCl in physiological solution) were also investigated in the present study. Transverse sections (15,pm thick and 60-100 from each animal) of the frozen segments of uterus were stained with glyoxylic acid (de la Torre & Surgeon, 1976) and viewed under a fluorescence microscope to visualize noradrenalinecontaining nerves. Hence, the presence of nerves in intact tissues or the effectiveness of denervation following injection of 6-hydroxydopamine was established for each preparation. Data from different groups were tested by analyses of variance (ANOVA). The effects of 6-hydroxydopamine were analyzed by comparing EC50 values to phenylephrine. Similar tests were used to determine the significance of responsiveness to maximal concentrations of phenylephrine and 100 mm potassium. The mean and standard error is given and a significance level of 0.05 was used in testing. (-)-Phenylephrine hydrochloride, 6-hydroxydopamine hydrobromide and (± )-propranolol hydrochloride were obtained from Sigma Chemical Company, U.S.A.
1500 g sec 0 I4 g
10.2 g
\I
/
