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Endopeptidase 24.11 inhibition does not modify uterotonic effects of endothelins in rat uterus
Peptides, Vol. 19, No. 9, pp. 1585–1593, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(98)00105-3
Endopeptidase 24.11 Inhibition Does Not Modify Uterotonic Effects of Endothelins in Rat Uterus MARIA KOUSIDES, MARGOT E. STORY1 AND JOCELYN N. PENNEFATHER Department of Pharmacology, Monash University, Clayton, Victoria, Australia, 3168 Received 6 March 1998; Accepted 15 June 1998 KOUSIDES M., M. E. STORY AND J. N. PENNEFATHER. Endopeptidase 24.11 inhibition does not modify uterotonic effects of endothelin in rat uterus. PEPTIDES 19(9) 1585–1593, 1998.—We investigated effects of the endopeptidase 24.11 inhibitor, SCH 39370, on uterotonic effects of endothelins (ETs) and sarafotoxin S6b. Responses of uteri from non-pregnant rats were inhibited by the ETA receptor antagonist, BQ123 (1 mM) but not the ETB receptor antagonist, BQ 788 (1 mM). ET-1, sarafotoxin S6b and ET-2 were more potent than ET-3 in tissues from non-pregnant and pregnant rats. SCH 39370 (10 mM) did not affect uterotonic responses to these peptides in either group, but inhibited those of big ET-1 in non-pregnant rat tissues, indicating inhibition of conversion of big ET-1 to ET-1. These data indicate that endopeptidase 24.11 does not inactivate the endothelin peptides in the rat uterus. © 1998 Elsevier Science Inc. Endothelin-1, -2, and -3 Sarafotoxin S6b Sarafotoxin S6c BQ 123 BQ 788 Big endothelin-1 Rat uterus Uterine stimulation Estrogen Pregnancy Endopeptidase 24.11 SCH 39370 Endothelin ETA receptors
THE 21-amino acid endothelin peptides ET-1, ET-2, and ET-3 are powerful stimulatory agonists at non-vascular as well as vascular smooth muscle (15,23). They can act upon either of two receptor subtypes, ETA and ETB to produce these effects (2,3,23). At the ETA receptor, ET-3 is less potent than ET-1 and ET-2, while at the ETB receptor all three isoforms are approximately equipotent. The precursor of endothelin-1, big endothelin-1, has little affinity for ET receptors (6), and its biologic activity is dependent upon its conversion to ET-1. Sarafotoxin S6b, which shares considerable sequence homology with the endothelin peptides, exhibits affinity for both ET receptor subtypes (1,39,40), whereas sarafotoxin S6c, in contrast, is highly selective for the ETB receptor. Although the physiological or pathological roles of endothelins in uterine function have yet to be established, immunoreactivity for endothelin-1 and big endothelin-1 are known to be present in uterine tissues (4,25,31,35). Levels of plasma and amniotic endothelin are known to increase as pregnancy progresses suggesting the possible participation of these peptides in the onset of labour (5,25). In the rat and
the human the enzyme endopeptidase 24.11, for which endothelin-1, but not sarafotoxin S6b, acts as a substrate (34,38) is present in the uterus. In the human endometrium its levels increase in the secretory phase of the menstrual cycle, when progesterone levels are high. In the rat uterus its levels are greatest in mid pregnancy (28) suggesting a role to constrain the uterotonic actions of substrate peptides during pregnancy. Uterotonic effects of endothelin peptides on the uterus of the non-pregnant rat are well-documented (3,19,30,32,37,41–43). The lower potency of endothelin-3 compared to that of endothelin-1 on non-gravid rat uterus has led to suggestions that the predominant receptor mediating endothelin-induced contraction in the non-pregnant rat was the ETA subtype, a hypothesis supported by the susceptibility of the endothelinmediated uterine contractions to blockade by the ETA receptor-selective antagonist, BQ-123 (16,30,37). The aims of the present study using tissues from nonpregnant, estrogen-primed virgin rats and from pregnant rats at Days 17–19 and Days 20 –21 of the 22-day pregnancy were three-fold. The first was to confirm that ETB
1 Requests for reprints should be addressed to Dr. M. E. Story, Department of Pharmacology, Monash University, Clayton, Victoria, 3168, Australia. E-mail: [email protected]
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receptor is not involved in mediating the uterotonic effects of the endothelin peptides in the uterus of the estrogenprimed rat. The second was to examine whether the uterotonic effects of the endothelin peptides were modulated by pregnancy, and the third was to determine whether there are any variations in sensitivity to inactivation by endopeptidase 24.11 as these might contribute to such modulation. To determine whether endopeptidase 24.11 inactivates endothelins in the uterus we have examined the influence of the endopeptidase 24.11 inhibitor, SCH 39370 (36), on the uterotonic potencies of endothelin-1, -2, and -3, and of sarafotoxin S6b; the last mentioned is resistant to degradation by endopeptidase 24.11 (34). We have previously shown that SCH 39370, like phosphoramidon, is effective in potentiating the uterotonic effects of those tachykinin peptides which are substrates for the enzyme (10,29). A further aim of the study was to examine the effects of SCH 39370 on uterine responses to big-endothelin-1, to determine whether this endopeptidase 24.11 inhibitor also affects the enzyme responsible for the conversion of big-endothelin-1 to endothelin-1. METHOD Animals and Tissue Preparations Ethical approval was obtained from the Monash University Standing Committee of Animal Ethics prior to any experimentation. Female virgin and pregnant Sprague–Dawley rats were housed at 22°C with a 12-h photoperiod cycle. They were allowed access to food and water ad libitum. Virgin rats (200 –260 g) were given a single injection of estradiol-17b-cypionate (20 mg/kg), SC, ,0.03 ml volume, 24 h prior to experimentation (29). On the day of experiment they were killed by a blow to the head followed by cervical dislocation. Prior to the removal of both uterine horns vaginal smears were taken to confirm the presence of cornified epithelial cells. The smears were fixed with 100% methanol and were allowed to air dry before staining with Giemsa stain to aid microscopic visualization of cells. Timed pregnant rats (240 –390 g), at various stages of gestation, were housed as described above. They were killed as outlined above at either Days 17–19 or Days 20 –21 of the 22 day pregnancy. Fetuses were killed by cervical dislocation after their removal from the uterine horns. After vaginal smears were taken from estrogen-primed animals a cut was made through the abdominal muscle layers to expose the uterus. Both uterine horns were removed and placed on a Petri dish containing modified Krebs–Henseleit solution of the following composition: (mM: NaCl, 118.0; KCl, 4.7; CaCl2.2H2O, 1.9; MgSO4.7H2O, 1.1; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.7). Surrounding fat and connective tissue were removed, and each horn bisected transversely to produce four preparations from each animal (29). Myometrial segments from pregnant animals were obtained as outlined above. Placental
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and fetal membranes were removed, and segments of uterus from fetus-bearing horns were obtained. Uterine preparations were tied onto tissue holders and placed into jacketed, siliconized, isolated organ baths (5 or 10 ml) containing bathing solution warmed to 37°C. The bathing solution was continuously bubbled with 95% O2 in CO2 to maintain pH at 7.4. Each myometrial preparation was attached to a Grass FTO3 isometric force transducer connected to a MacLab recording instrument. All preparations were set up so that longitudinally arranged muscle fibers were orientated vertically. Tissues were allowed to equilibrate in modified Krebs–Henseleit solution for 30 min under an initial force of 1 g (non-gravid rats) or 2 g (gravid animals). The bathing solution was replaced three to four times during this period. Agonist Effects Discrete log concentration-response curves were obtained using one-half log-unit concentration increments. Preparations were exposed to each agonist concentration for 4 min, then washed with 2–3 replacements of the bath volume; 20 min elapsed before the next concentration was tested. Only one log concentration-response curve was obtained on each preparation. Each preparation was exposed to 40 mM KCl solution (mM: NaCl, 82.7; KCl, 40.0; CaCl2.2H20, 2.5; MgSO4.7H2O, 0.5; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.7) 20 – 40 min after the last concentration of peptide was washed from the baths to confirm tissue viability and the selectivity of antagonists and the enzyme inhibitor used. Endothelin Receptor Blockade Uterine preparations were exposed to the ETA receptor antagonist, BQ-123 (1 mM; Reference 16) or the ETB receptor antagonist, BQ-788 (1 mM; Reference 17) for 30 min following the initial equilibration period. Log concentration–response curves were then constructed to endothelin-1 or sarafotoxin S6b. The antagonists were replaced each time the bath was washed, and so remained in contact with the tissues throughout the experiment. Inhibition of Endopeptidase 24.11 SCH 39370 (10 mM) was used to inhibit endopeptidase 24.11 (36). It was added to the bath for 30 min after the initial equilibration period, and replaced each time the bath was washed during the construction of log concentrationresponse curves to endothelin-1, endothelin-2, endothelin-3, sarafotoxin S6b, and sarafotoxin S6c. In a separate series of experiments, the ability of SCH 39370 to inhibit responses to the endothelin-1 precursor, big endothelin-1, was investigated. Uterine preparations from estrogen-treated rats were exposed to carbachol (10 mM) twice, in order to obtain reproducible responses, using a similar protocol as that outlined above, except that tissues were exposed to agonist concentrations for 5 min. If the two
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FIG. 1. Representative traces showing contractions evoked by endothelin-1 (upper panel) and sarafotoxin S6c (lower panel) on myometrial preparations obtained from an estrogen-treated rat, and also to a bathing solution in which 40 mM KCl replaced 40 mM NaCl. Agonist addition is shown by arrows; W indicates a wash. Preparations were placed under an initial force of 1g. ET-1 evoked concentration-related contractions. Sarafotoxin S6c was inactive. Exposure to 40 mM KCl produced contractile responses of both tissues.
carbachol-evoked contractions differed substantially, a third application of carbachol was employed. After 15–20 min and extensive washing, a contractile response to either endothelin-1 (5 nM) or big endothelin-1 (12 nM) was obtained. After drug washout, SCH 39370 (10 mM) was added to some of the preparations, others acted as time controls. 30 min later, the contractile response to the original peptide was reassessed. Data Analysis Responses to agonists were measured as area under the force-time curve (g.s) over the 4-min period of agonist exposure and were normalized for tissue weight. Since in most instances the highest concentration of agonist that could be used did not produce maximal tissue responses it was not usually possible to calculate EC50 values for agonists. The relative potencies of the agonists used, however, and the effects of endothelin receptor antagonists and SCH 39370 on log concentration-response curves to agonists were examined using linear regression analysis of data points lying approximately between 15– 85% of the greatest response obtained, for estimation of potency ratios with 95% confidence limits. Fitted regressions were tested for linearity, parallelism and coincidence using procedures outlined in Documenta Geigy (8). The positions of pairs of lines meeting the criteria of linearity and parallelism were deemed to differ when the 95% confidence limits of potency ratios did not include one (26). The ratios of potency of the agonist in the absence and presence of an antagonist were then used to estimate the apparent pKB using the formula described by Furchgott (12) viz. log [antagonist concentration/potency ratio minus one]. Analyses of variance (followed by Tukey’s test) and Student’s paired or unpaired t tests were also used, p , 0.05 being the criterion for acceptance of statistical significance; ‘n’ refers to the number of animals.
Drugs Endothelin-1 (human, porcine) was obtained from Auspep or the American Peptide Company; endothelin-2 (human), endothelin-3 (human, rat), sarafotoxin S6b, sarafotoxin S6c and BQ-123 [cyclo(D-Asp-L-Pro-D-Val-L-Leu-D-Trp)] were obtained from American Peptide Company. Big endothelin-1 was obtained from Auspep. BQ-788 (Dmpc-g-MeLeu9-D-Trp(l-CO2CH3)-D-Nle-OH) was purchased from Novabiochem. Endothelin-1 (Auspep) and sarafotoxin S6b were made up in distilled H2O. The remaining peptides were first dissolved in 200 ml dimethyl sulphoxide (DMSO) before being made up to the required concentrations (10 or 100 mM for agonists; 1 mM for antagonists) in distilled H2O. The highest concentration of DMSO in the organ baths was 0.002%. SCH 39370 (N-[N-[1-(S)-carboxyl-3phenylpropyl]-(S)-phenylalanyl]-(S)-isoserine) was made up to a 1 mM stock solution in 0.1% NaHCO3. Myometrial activity was not influenced by either vehicle. Estradiol cypionate was dissolved in a small volume of 100% ethanol and then made up to 20 mg/ml in peanut oil; this solution was kept in the dark. RESULTS Tissues from Estrogen-primed Animals Agonist potency order . Endothelins -1, -2, and -3, and sarafotoxin S6b, produced concentration-dependent contractile responses of myometrium from estrogen-primed, non-pregnant rats. These were characterized by an increase in the frequency of spontaneous contractions arising from the longitudinally arranged smooth muscle layer at concentrations typically up to 1–3 nM, and a superimposed increase in basal force at higher concentrations. Figure 1 shows typical traces obtained in response to ET-1 and 40 mM KCl, whereas sarafotoxin S6c was ineffective. Endothelin-1 induces concentration-related uterotonic activity
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at concentrations of 3 nM; contractions are not maximal at 0.1 mM. In contrast, sarafotoxin S6b clearly produced maximal responses at this concentration (Fig. 2). The order of agonist potency was sarafotoxin S6b . endothelin-1 5 endothelin-2 . endothelin-3 . sarafotoxin S6c (Fig. 2). The mean log concentration-response curve to sarafotoxin S6b lay almost four-fold to the left of that to endothelin-1 (potency ratio 5 3.68; 95% confidence limits 5 1.51, 10.37; df 5 21) whereas endothelin-3 was 8.5 times less potent (potency ratio endothelin-1 to endothelin3 5 8.50; 95% confidence limits 5 4.22, 22.43; df 5 39). Sarafotoxin S6c lacked uterotonic activity in concentrations of up to 0.1 mM in all four preparations in which it was tested (Fig. 1 and 2).
FIG. 2. Graph showing mean log concentration–response curves constructed using preparations of uterus from non-pregnant rats to endothelin-1 (E), endothelin-2 (M), endothelin-3 (‚), sarafotoxin S6b (F) and sarafotoxin S6c (f). Vertical axis represents the area under the force-time curve, normalized for tissue weight. Responses are shown as mean, n 5 4 – 6; vertical bars represent SEM.
Effects of Endothelin Antagonists Responses to endothelin-1 and sarafotoxin S6b were significantly inhibited by the ETA receptor antagonist BQ-123 (1 mM; Figs. 3A & B). The magnitude of the rightward shifts of the concentration-response curves to these peptides in the presence of BQ-123 were 20.23-fold for endothelin-1 (95% confidence limits 5 11.24, 47.53; df 5 20) and 35.99-fold
FIG. 3. Log concentration–response curves of uterus from non-pregnant rats to endothelin-1 or sarafotoxin S6b in the absence (E) or presence of BQ-123 (1 mM; F; A and B, respectively) or BQ-788 (1 mM; F; C and D, respectively). Responses are shown as mean; vertical bars represent SEM; n 5 4 – 6. *p , 0.05, ***p , 0.001, compared to corresponding point of control curve (Tukey’s test). Peptide-evoked contractions were attenuated by BQ-123 but not by BQ-788.
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TABLE 1 POTENCY RATIOS OBTAINED FROM LINEAR REGRESSION ANALYSIS OF LOG CONCENTRATION-RESPONSE CURVES CONSTRUCTED IN TISSUES FROM NON-PREGNANT RATS IN THE ABSENCE AND PRESENCE OF SCH 39370 (10 mM) Peptide
Number of Tissues
Potency Ratio
95% Confidence Limits (df)
Endothelin-1 Endothelin-2 Endothelin-3 Sarafotoxin S6b Sarafotoxin S6c
6 6 5 5 3
1.33 1.56 1.31 1.16 *
0.55, 3.89 (38) 0.91, 2.77 (43) 0.77, 2.34 (31) 0.61, 2.23 (26) *
* Indicates not determined. In each case, confidence limits included 1.0, indicating no significant effect of SCH 39370.
for sarafotoxin S6b (95% confidence limits 5 15.98, 131.23; df 5 20). pKB values calculated from these shifts were of 7.23 and 7.54, respectively, for the two peptides. The ETB receptor antagonist, BQ-788 (1 mM) had no effect upon log concentration-response curves to endothelin-1 or sarafotoxin S6b (Figs. 3C & D). Effects of SCH 39370 Experiments were conducted concurrently with the experiments yielding the control curves shown in Fig. 2. The endopeptidase 24.11 inhibitor, SCH 39370 (10 mM), had no effect upon responses of myometrium to the peptides (Table 1). In a separate series of experiments, uterine preparations from six estrogen-treated rats were exposed to carbachol (10 mM). All preparations responded consistently (n 5 6; p . 0.05). The mean response was 7.08 6 0.45 g.s/mg tissue (n 5 24 preparations from 6 animals). The subsequent addition of ET-1 (5 nM) caused contractions of 4.53 6 1.07 g.s/mg (Fig. 4). Responses to big endothelin-1 (12 nM) were significantly lower (p , 0.05) than those obtained to endothelin-1, giving values of 1.88 6 0.53 g.s/mg tissue SCH 39370 was without effect on responses to ET-1, but significantly reduced those to big ET-1 (Fig. 4). Tissues from Pregnant Animals Agonist potency order . All preparations responded similarly to the modified KCl bathing solution. The contractures induced were evident immediately and were usually sustained for the duration of measurement. Previous exposure to a peptide agonist produced no difference in the magnitude of responses elicited by subsequent exposure to the high potassium solution. The relative orders of agonist potencies were similar at Days 17–19 and at Days 20 –21 (Fig. 5). Thus the potency order in tissues from both groups of animals was sarafotoxin S6b 5 ET-1$ ET-2 . ET-3. Sarafotoxin S6c was not investigated. Interestingly, the 1og concentration effect curves to sarafotoxin S6b in tissues
FIG. 4. Histograms showing the effects of ET-1 and big ET-1 on preparations of myometrium from estrogen-primed rats before and after exposure to SCH 39370 (10 mM). The vertical axis represents the area under the force-time curve, normalized for tissue weight. Each column represents the mean six experiments; vertical bars represent SEM. *p , 0.05, Student’s paired t-test.
from pregnant animals did not plateau at the highest concentrations used (Fig. 5) as they did in tissues from nonpregnant estrogen-primed animals (Fig. 2). The potency of this peptide was similar to rather than greater than that of ET-1, as was the case in tissues from the non-pregnant animals (Table 2). Furthermore, responses to the maximal concentrations of each peptide used, namely 0.1 mM, when normalized for tissue weight, were similar at the two stages of pregnancy (Fig. 5) and these responses were generally similar to those observed in tissues from non-pregnant animals (ANOVA, p . 0.05). Effects of SCH 39370 The effects of SCH 39370 (10 mM) on responses to the peptides in preparations from pregnant rats are shown in Figs. 6 and 7. Responses of myometrium from day 17–19 pregnant rats to endothelins-1 and -3 and to sarafotoxin S6b were not modified by SCH 39370, but there was some enhancement of the effects of endothelin-2 (Fig. 6). In this set of experiments, but in no others, there was also an enhancement by SCH 39370 of the effects of high potassium solution indicating that the effect on endothelin-2 was not selective. On myometrium from D20 –21 pregnant rats, the mean concentration-response curves of the four peptides were unaltered in the presence of SCH 39370 (Fig. 7). DISCUSSION The major, albeit unexpected, finding of this study was that the enzyme endopeptidase 24.11 does not limit the uterotonic effects of endothelins in uterine preparations from
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FIG. 5. Graphs showing mean log concentration–response curves to endothelin-1 (E), endothelin-2 (M), endothelin-3 (‚), and sarafotoxin S6b (F) in tissues taken from rats at Days 17–19 (D17–19) and Days 20 –21 (D20 –21) of pregnancy. Vertical axes represent the area under the force–time curve, normalized for tissue weight. Each point represents the mean of 4 – 6 experiments; vertical bars represent SEM.
estrogen-primed, non-pregnant rats or from late pregnant rats. The study also indicated that the endopeptidase 24.11 inhibitor used in this study, SCH 39370, may inhibit the conversion of big endothelin-1 to endothelin-1 in the uterus of the estrogen-primed, non-pregnant rat. The present study has also 1) confirmed that the endothelins and sarafotoxin S6b contract longitudinally-arranged smooth muscle of uterine horns from estrogen-primed rats and late pregnant rats by activating ETA receptors and 2) provided strong evidence that ETB receptors do not contribute to the actions of the peptides in tissues from the estro-
TABLE 2 POTENCY RATIOS OBTAINED FROM LINEAR REGRESSION ANALYSIS OF LOG CONCENTRATION RESPONSE CURVES CONSTRUCTED FOR ET-2, ET-3, AND SARAFOTOXIN S6B COMPARED TO CORRESPONDING CURVES FOR ET-1 IN PREPARATIONS FROM ESTROGEN PRIMED AND PREGNANT RATS Potency Ratio (95% CL; df)* Peptide Endothelin-2 Endothelin-3 Sarafotoxin S6b
Estrogen Treated
Pregnant: D17–19
Pregnant: D20–21
1.68 (0.88, 3.40; 44) 0.12† (0.05, 0.24; 39) 3.68† (1.51, 10.37; 21)
0.28† (0.07, 0.71; 28) 0.04† (0.02, 0.06; 12) 1.09 (0.61, 1.95; 25)
0.55 (0.19, 1.40; 33) 0.05† (0.02, 0.10; 24) 0.62 (0.17, 1.83; df)
* 95% confidence limits and degrees of freedom (df). † Indicates a significant difference in the location of the mean log concentrationresponse curves to that peptide relative to endothelin-1 (i.e., confidence limits do not include 1.0). A potency value greater than one indicates that the log concentrationresponse curve lay to the left of that for ET-1.
FIG. 6. Log concentration–response curves constructed using uterus taken from rats at Days 17–19 of pregnancy to ET-1, ET-2, ET-3, or sarafotoxin S6b in the absence (open symbols) or presence of SCH 39370 (10 mM; filled symbols). Responses are shown as mean of four experiments, vertical bars represent SEM. *p , 0.05 compared to corresponding point of control curve.
ENDOTHELINS AND EC 24.11 IN RAT UTERUS
FIG. 7. Log concentration–response curves constructed using uterus taken from rats at Days 20 –21 of pregnancy to ET-1, ET-2, ET-3, or sarafotoxin S6b in the absence (open symbols) or presence of SCH 39370 (10 mM; filled symbols). Responses are means of six experiments; vertical bars represent SEM.
gen-primed group. Our confirmation of earlier work (30,37) that ETA but not ETB receptors mediate endothelin-induced contractions of rat longitudinal myometrium of estrogenprimed, non-pregnant rats is based on findings that (1) endothelin-3 was less potent as a uterotonic agent than endothelins -1, -2, and sarafotoxin S6b; (2) sarafotoxin S6c, which selectively activates ETB receptors (40), was ineffective, (3) the ETA -selective antagonist, BQ-123 (16) produced apparently competitive inhibition of responses of myometrium to endothelin-1 and sarafotoxin S6b, and (4) responses to the latter peptides were unaltered by the presence of the ETB-selective antagonist, BQ-788 (17). The apparent pKB value we obtained for BQ-123 versus endothelin-1 (7.23) is similar to the pA2 value of this antagonist reported by Rae et al. (30) for rat uterus. Our findings are of additional interest given that the concentrations of estrogen we used to prime animals were some ten times lower than those used in the earlier experiments. It is conceivable that sarafotoxin S6b may activate binding sites distinct from or additional to those activated by endothelin-1. We found, however, that BQ-123 inhibited the responses to both peptides on rat longitudinally-oriented
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myometrium to a similar extent, indicating that in this tissue these peptides bind to the same sites. The apparent pKB values of BQ-123 as an antagonist of endothelin-1 and sarafotoxin S6b on rat myometrium were comparable with those estimated previously with tissues from non-pregnant rat uterus (37), and in human myometrium (13). The relative order of agonist potency of the endothelin isopeptides on days 17–19 and days 20 –21 indicated that the ETA receptor mediates the effects of their contractile effects on the pregnant uterus. The response to KCl and the responses obtained to the maximum concentrations of the peptides were apparently greater in tissues from pregnant rats, however this increased responsiveness paralleled the considerable uterine hypertrophy characteristic of pregnancy. Thus responses of tissues from gravid rats, when normalized for tissue weight, did not exceed those obtained in tissues from non-pregnant animals. Other authors have reported an increased potency of endothelin-1 on rat gravid uterine preparations (3,32). These authors, however, expressed their data as absolute force, as area under the force-time curve, or as a percentage of the response to KCl. All of these measures are influenced by uterine hypertrophy. Yallampalli and Garfield (43) reported an increased ability of endothelin-1 to contract myometrial strips obtained from delivering rats (D22 of gestation) compared to preparations obtained from near-term pregnant rats (D18). This suggests a possible role for endothelins in labour. The present experiments did not address this possibility. Endothelin degradation can be effected by neutral endopeptidase 24.11 (EC 24.11). This is a multisubstrate (14,28,22) membranous metalloprotease which cleaves endothelin-1 preferentially between Leu5-Ser6; this is then followed by cleavage at the carboxy-terminus tail (34,38). Amino acid substitutions at the corresponding positions on endothelins-2 and -3 confer resistance to EC 24.11 at this site but promote cleavage at the tail. In the rat, the activity of myometrial EC 24.11 increases markedly during gestation, declines slightly just prior to parturition, and continues to fall post-partum (28). Hence, modulation of the activity of EC 24.11 offers a potentially significant mechanism for regulating the in vivo concentrations of endothelins. In contrast to the endothelins, the sarafotoxins are relatively resistant to degradation by EC 24.11 (34). Thus these peptides are useful tools in investigations of the functional significance of EC 24.11-mediated endothelin degradation. In tissues from pregnant animals, the potency of sarafotoxin S6b relative to that of endothelin-1 was reduced rather than increased. We are unable to offer an explanation for this decrease in relative potency, but it indicates that there are no pregnancy-related decreases in the susceptibility of the endothelin peptides to degradation by endopeptidase 24.11. Generally, inhibition of this enzyme by SCH 39370 had negligible effect on the potencies of any of the peptides in tissues from either non-pregnant or pregnant animals. An
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exception was seen in tissues taken from rats on Days 17–19 of pregnancy in which SCH 39370 enhanced responses to some concentrations of ET-2. In this group of experiments it also enhanced responses to high potassium. It is unlikely that the concentration (10 mM) of SCH 39370 we used to inhibit this enzyme are too low, as at 3 mM, it produces maximal potentiation of the effects of neurokinin A (10). The latter peptide is a good substrate for EC 24.11 (14). The possibility exists that EC 24.11 may not be the main contributor to endothelin degradation in the uterus. Fagny et al. (9) have described the ability of soy bean tryptase inhibitor, an inhibitor of serine proteases, including cathepsin G, to inhibit the appearance of [125I]-endothelin-1 degradation products from activated polymorphonuclear neutrophils. In their experiments, an EC 24.11 inhibitor, phosphoramidon, was devoid of any such effect. An interesting possibility is that cathepsin G rather than EC 24.11 may be involved in endothelin degradation in the uterus. A novel finding of this study was that SCH 39370 decreased the response of the uterine preparations from non-pregnant rats to big ET-1. As already described, SCH 39370 did not modify the endothelin-1-evoked contraction. SCH 39370 has previously been reported to be a selective inhibitor of EC 24.11 (36). Other EC 24.11 inhibitors, such as phosphoramidon (11,22) and CGS 26303 (S)-2-biphe-
nyl-4-yl-1-(iH-tetrazol-5-yl)-ethylamino-methyl phosphonic acid (7) also function as ECE inhibitors (30). In the absence of biochemical data it is premature to postulate whether the effect of SCH 39370 on the response to big ET-1 occurs through the inhibition of ECE or of another enzyme involved in the conversion of big ETs to endothelins. However, it is likely that administration of SCH 39370 would limit rather than exacerbate the involvement of endogenous endothelins in uterine contractility. In conclusion, this study has confirmed that the endothelins are potent stimulators of uterine contractility of tissues from pregnant as well as from non-pregnant rats. In this species, in contrast to the human (13,27) and the guinea-pig (18) their uterotonic effects are mediated exclusively by ETA receptors in both pregnant and non-pregnant states. Their effects are not significantly influenced by inhibition of EC 24.11, suggesting that this endopeptidase is not a significant determinant of endothelin potency in this tissue in this species, even during pregnancy when the levels of this enzyme are reported to increase (28). ACKNOWLEDGEMENTS We are indebted to Dr. E. Sybertz, for a generous gift of SCH 39370.
REFERENCES 1. Ambar, I.; Kloog, Y.; Schvartz, I.; Hazum, E.; Sokolovsky, M. Competitive interaction between endothelin and sarafotoxin: binding and phosphoinositides hydrolysis in rat atria and brain. Biochem. Biophys. Res Commun. 158:195–201;1989. 2. Arai, H.; Hori S.; Aramori, I.; Ohkubo, H.; Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature (London). 348:730 –732; 1990. 3. Calixto, J. B.; Rae, G. A. Effects of endothelins, Bay K8644 and other oxytocics in non-pregnant and late pregnant rat isolated uterus. Eur. J. Pharmacol. 192:109 –116; 1991. 4. Cameron, I. T.; Bacon, C. R.; Collett, G. P.; Davenport, A. P. Endothelin expression in the uterus. J. Steroid. Biochem. Mol. Biol. 53:209 –214; 1995. 5. Chou, J.; Wang, Y. N.; Chang, D.; Chang, J. K.; Avila, C.; Romero, R. Big endothelin in plasma and amniotic fluid. J. Cardiovasc. Pharmacol. 17(Suppl. 7):S430 –S433; 1991. 6. Davenport, A. P.; Cameron, I. T.; Smith, S. K.; Brown, M. J. Binding sites for iodinated ET-1, ET-2 and ET-3 demonstrated on human uterine glandular epithelial cells by quantitative high-resolution autoradiography. J. Endocrinol. 129:149 –154; 1991. 7. De Lombaert, S.; Ghai, R. D.; Jeng, A. Y.; Trapani, A. J.; Webb, R. L. Pharmacological profile of a non-peptidic dual inhibitor of neutral endopeptidase 24.11 and endothelin converting enzyme. Biochem. Biophys. Res. Commun. 204:407– 412; 1994. 8. Geigy Scientific Tables, Vol 2, 8th edn. Introduction to statistics, statistical tables, mathematical formulae, ed Lerner. Ciba–Geigy Ltd Basle, Switzerland, 1982, pp 214. 9. Fagny, C.; Michel, A.; Nortier, J.; Deschodt–Lanckman, M. Enzymatic degradation of endothelin-1 by activated human polymorphonuclear neutrophils. Regul. Pept. 42:27–37; 1992.
10. Fisher, L.; Pennefather, J. N. Potencies of agonists acting at tachykinin receptors in the estrogen-primed rat uterus: effects of peptidase inhibitors. Eur. J. Pharmacol., 335:221–226; 1997. 11. Fukuroda, T.; Noguchi, K.; Tsuchida, S.; Nishikibe, M; Ikemoto, F.; Okada, K.; Yano, M. Inhibition of biological actions of big endothelin-1 by phosphoramidon. Biochem. Biophys. Res. Commun. 172:390 –395; 1990. 12. Furchgott, R. F. The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory. In: Blaschko, H.; Muscholl, E., eds. Handbook of Experimental Pharmacology. Springer–Verlag: Berlin, vol. 33; 283–336; 1972. 13. He´luy, V.; Germain, G.; Fournier, T.; Ferre´, F.; Breuiller– Fouche´, M. Endothelin ETA receptors mediate human uterine smooth muscle contraction. Eur. J. Pharmacol. 285:89 –94; 1995. 14. Hooper, N. M.; Kenny, J; Turner, A. J. The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase (angiotensin-converting enzyme). Biochem. J. 231:357–361; 1985. 15. Huggins, J. P.; Pelton, J. T.; Miller, R. C. The structure and specificity of endothelin receptors: their importance in physiology and medicine. Pharmacol. Ther. 59, 55–123; 1993. 16. Ihara, M.; Noguchi, K.; Saeki, T.; Fukuroda, T.; Tsuchida, S.; Kimura, S.; Fukami, T.; Ishikawa, K.; Nishikibe, M.; Yano, M. Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor. Life Sci. 50:247–255; 1992. 17. Ishikawa, K.; Ihara, M.; Noguchi, K.; Mase, T.; Mino, N.; Saiki, T.; Fukuroda, T.; Fukami, T.; Ozaki, S.; Nagase, T.; Nishikibe, M.; Yano, M. Biochemical and pharmacological
ENDOTHELINS AND EC 24.11 IN RAT UTERUS
18.
19.
20.
22.
23. 24.
25.
26. 27.
28. 29. 30.
31.
profile of a potent and selective endothelinB-receptor antagonist, BQ-788. Proc. Natl. Acad. Sci. USA. 91:4892– 4896; 1994. Kousides, M.; Story, M. E.; Pennefather, J. N. Endothelin receptors mediating contractile response of rat and guinea-pig uterus. Proc. Aust. Soc. Clin. Exp. Pharmacol. Toxicol. 2, 51; 1995. Kozuka, M.; Ito, T.; Hirose, S.; Eguchi, S.; Hagiwara, H. Endothelin action on rat uterus is inhibited by an inhibitor of protein kinase C and by inhibitors of the phospholipase A2arachidonic acid-lipoxygenase pathway. Biomed. Res. 11:287–289; 1990. Kozuka, M.; Ito, T.; Hirose, S.; Takahashi, K.; Hagiwara, H. Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channel. Biochem. Biophys. Res. Commun. 159, 317–323; 1989. Malfroy, B.; Schwartz, J.C. Properties of ‘enkephalinase’ from rat kidney: comparison of dipeptidyl-carboxypeptidase and endopeptidase activities. Biochem. Biophys. Res. Commun. 106:276 –285; 1982. Masaki, T.; Vane J. R.; Vanhoutte, P. M. International union of pharmacology nomenclature of endothelin receptors. Pharmacol. Rev. 46, 137–142;1994. Nisell, H.; Hemse´n, A.; Lunell, N. O.; Wolff, K.; Lundberg, M. J. Maternal and fetal levels of a novel polypeptide, endothelin: evidence for release during pregnancy and delivery. Gynecol. Obstet. Invest. 30:129 –132; 1990. Ohbuchi, H.; Nagai, K.; Yamaguchi, M.; Ikenoue, T.; Mori, N.; Kitamura, K.; Araki, S.; Toshimori, K. Endothelin-1 and big endothelin-1 increase in human endometrium during menstruation. Am. J. Obstet. Gynecol. 173:1483–1490; 1995. Orre, M.; Pennefather, J. N.; Story, M. E.; Haynes, J. M. The effects of P2 purinoceptor agonists on the isolated portal vein of the guinea pig. Eur. J. Pharmacol. 316:229 –236; 1996. Osada, K.; Tsunoda, H.; Miyauchi, Y.; Sugishita, Y.; Kubo, T.; Goto, K. Pregnancy increases ET-1-induced contraction and changes receptor subtypes in uterine smooth muscle in humans Am. J. Physiol. 272:R541–548; 1997. Ottlecz, A.; Walker, S.; Conrad M.; Starcher, B. Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus, J. Cell. Biochem. 45:401– 411; 1991. Pennefather, J. N.; Zeng, X. -P.; Gould, D.; Hall, S.; Burcher, E. Mammalian tachykinins stimulate rat uterus by activating NK-2 receptors. Peptides. 14:169 –174; 1993. Rae, G. A.; Calixto, J. B.; D’Orle´ans–Juste, P. Big-endothelin-1 contracts rat isolated uterus via a phosphoramidon-sensitive endothelin ETA receptor-mediated mechanism. Eur. J. Pharmacol. 240:113–119; 1993. Riley, S. C.; Butt, A. R.; Doughton, B. W.; Li, S. Y.; Zheng,
1593
32. 33.
34.
35.
36.
37. 38.
39.
40.
41.
42. 43.
S. H.; Findlay, J. K.; Salamonsen, L. A. Endothelin in the ovine uterus during the oestrous cycle and early pregnancy. J. Reprod. Fertil. 100:451– 459; 1994. Sakata, K.; Karaki, H. Effects of endothelin on cytosolic Ca21 level and mechanical activity in rat uterine smooth muscle. Eur. J. Pharmacol. 221:9 –15; 1992. Sakurai, T; Yanagisawa, M; Takuwa, Y; Miyazaki, H.; Kinura, S.; Goto, K.; Masaki, T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of endothelin receptor. Nature (London). 348, 732–735; 1990. Sokolovsky, M.; Galron, R.; Kloog, Y.; Bdolah, A.; Indig, F. E.; Blumberg, S.; Fleminger, G. Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance. Proc. Natl. Acad. Sci. USA. 87, 4702– 4706; 1990. Svane D.; Larsson, B.; Alm, P.; Andersson, K. E.; Forman, A. Endothelin-1: immunocytochemistry, localization of binding sites, and contractile effects in human uteroplacental smooth muscle. Am. J. Obstet. Gynecol. 168, 233–241; 1993. Sybertz, E. J.; Chiu, P. J. S.; Vemulapalli, S.; Pitts, B.; Foster, C. J.; Watkins, R. W.; Barnett, A.; Haslanger, M. F. SCH 39370, a neutral metalloendopeptidase inhibitor, potentiates biological responses to atrial natriuretic factor and lowers blood pressure in desoxycortisone acetate-sodium hypertensive rats. J. Pharmacol. Exp. Ther. 250:624 – 631; 1989. Tsunoda, H.; Miyauchi, T.; Fujita, K.; Kubo, T.; Goto, K. Mechanism of rat uterine smooth muscle contraction induced by endothelin-1. Br. J. Pharmacol. 110:1437–1446; 1993. Vijayaraghavan, J.; Scicli, A. G.; Carretero, O. A.; Slaughter, C.; Moomaw, C.; Hersh, L. B. The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase). J. Biol. Chem. 265:14150 –14155; 1990. Williams, D. L.; Jones, K. L.; Pettibone, D. J.; Lis, E. V.; Clineschmidt, B. V. Sarafotoxin S6c, an agonist which distinguishes between endothelin receptor subtypes. Biochem. Biophys. Res. Commun. 175, 556 –561; 1991b. Williams, D. L.; Jones, K. L.; Colton, C. D.; Nutt, R. F. Identification of high affinity endothelin-1 receptor subtypes in human tissues. Biochem. Biophys. Res. Commun. 180:475– 480; 1991a. Wollberg, Z.; Bousso–Mittler, D.; Bdolah, A.; Kloog, Y.; Kochva, E.; Sokolovsky, M. Endothelins and sarafotoxins: effects on motility, binding properties and phosphoinositide hydrolysis during the estrous cycle of the rat uterus. J. Basic Clin. Physiol. Pharmacol. 3:41–57; 1992a. Wollberg, Z.; Shinnar, N.; Bdolah, A.; Kochva, E. Endothelin and sarafotoxin: influence on steroid-regulated motility of rat uterus. Life Sci. 51, L57– 60; 1992b. Yallampalli, C.; Garfield, R. E. Uterine contractile responses to endothelin-1 and endothelin receptors are elevated during labor. Biol. Reprod. 51:640 – 645; 1994.
PII S0196-9781(98)00105-3
Endopeptidase 24.11 Inhibition Does Not Modify Uterotonic Effects of Endothelins in Rat Uterus MARIA KOUSIDES, MARGOT E. STORY1 AND JOCELYN N. PENNEFATHER Department of Pharmacology, Monash University, Clayton, Victoria, Australia, 3168 Received 6 March 1998; Accepted 15 June 1998 KOUSIDES M., M. E. STORY AND J. N. PENNEFATHER. Endopeptidase 24.11 inhibition does not modify uterotonic effects of endothelin in rat uterus. PEPTIDES 19(9) 1585–1593, 1998.—We investigated effects of the endopeptidase 24.11 inhibitor, SCH 39370, on uterotonic effects of endothelins (ETs) and sarafotoxin S6b. Responses of uteri from non-pregnant rats were inhibited by the ETA receptor antagonist, BQ123 (1 mM) but not the ETB receptor antagonist, BQ 788 (1 mM). ET-1, sarafotoxin S6b and ET-2 were more potent than ET-3 in tissues from non-pregnant and pregnant rats. SCH 39370 (10 mM) did not affect uterotonic responses to these peptides in either group, but inhibited those of big ET-1 in non-pregnant rat tissues, indicating inhibition of conversion of big ET-1 to ET-1. These data indicate that endopeptidase 24.11 does not inactivate the endothelin peptides in the rat uterus. © 1998 Elsevier Science Inc. Endothelin-1, -2, and -3 Sarafotoxin S6b Sarafotoxin S6c BQ 123 BQ 788 Big endothelin-1 Rat uterus Uterine stimulation Estrogen Pregnancy Endopeptidase 24.11 SCH 39370 Endothelin ETA receptors
THE 21-amino acid endothelin peptides ET-1, ET-2, and ET-3 are powerful stimulatory agonists at non-vascular as well as vascular smooth muscle (15,23). They can act upon either of two receptor subtypes, ETA and ETB to produce these effects (2,3,23). At the ETA receptor, ET-3 is less potent than ET-1 and ET-2, while at the ETB receptor all three isoforms are approximately equipotent. The precursor of endothelin-1, big endothelin-1, has little affinity for ET receptors (6), and its biologic activity is dependent upon its conversion to ET-1. Sarafotoxin S6b, which shares considerable sequence homology with the endothelin peptides, exhibits affinity for both ET receptor subtypes (1,39,40), whereas sarafotoxin S6c, in contrast, is highly selective for the ETB receptor. Although the physiological or pathological roles of endothelins in uterine function have yet to be established, immunoreactivity for endothelin-1 and big endothelin-1 are known to be present in uterine tissues (4,25,31,35). Levels of plasma and amniotic endothelin are known to increase as pregnancy progresses suggesting the possible participation of these peptides in the onset of labour (5,25). In the rat and
the human the enzyme endopeptidase 24.11, for which endothelin-1, but not sarafotoxin S6b, acts as a substrate (34,38) is present in the uterus. In the human endometrium its levels increase in the secretory phase of the menstrual cycle, when progesterone levels are high. In the rat uterus its levels are greatest in mid pregnancy (28) suggesting a role to constrain the uterotonic actions of substrate peptides during pregnancy. Uterotonic effects of endothelin peptides on the uterus of the non-pregnant rat are well-documented (3,19,30,32,37,41–43). The lower potency of endothelin-3 compared to that of endothelin-1 on non-gravid rat uterus has led to suggestions that the predominant receptor mediating endothelin-induced contraction in the non-pregnant rat was the ETA subtype, a hypothesis supported by the susceptibility of the endothelinmediated uterine contractions to blockade by the ETA receptor-selective antagonist, BQ-123 (16,30,37). The aims of the present study using tissues from nonpregnant, estrogen-primed virgin rats and from pregnant rats at Days 17–19 and Days 20 –21 of the 22-day pregnancy were three-fold. The first was to confirm that ETB
1 Requests for reprints should be addressed to Dr. M. E. Story, Department of Pharmacology, Monash University, Clayton, Victoria, 3168, Australia. E-mail: [email protected]
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receptor is not involved in mediating the uterotonic effects of the endothelin peptides in the uterus of the estrogenprimed rat. The second was to examine whether the uterotonic effects of the endothelin peptides were modulated by pregnancy, and the third was to determine whether there are any variations in sensitivity to inactivation by endopeptidase 24.11 as these might contribute to such modulation. To determine whether endopeptidase 24.11 inactivates endothelins in the uterus we have examined the influence of the endopeptidase 24.11 inhibitor, SCH 39370 (36), on the uterotonic potencies of endothelin-1, -2, and -3, and of sarafotoxin S6b; the last mentioned is resistant to degradation by endopeptidase 24.11 (34). We have previously shown that SCH 39370, like phosphoramidon, is effective in potentiating the uterotonic effects of those tachykinin peptides which are substrates for the enzyme (10,29). A further aim of the study was to examine the effects of SCH 39370 on uterine responses to big-endothelin-1, to determine whether this endopeptidase 24.11 inhibitor also affects the enzyme responsible for the conversion of big-endothelin-1 to endothelin-1. METHOD Animals and Tissue Preparations Ethical approval was obtained from the Monash University Standing Committee of Animal Ethics prior to any experimentation. Female virgin and pregnant Sprague–Dawley rats were housed at 22°C with a 12-h photoperiod cycle. They were allowed access to food and water ad libitum. Virgin rats (200 –260 g) were given a single injection of estradiol-17b-cypionate (20 mg/kg), SC, ,0.03 ml volume, 24 h prior to experimentation (29). On the day of experiment they were killed by a blow to the head followed by cervical dislocation. Prior to the removal of both uterine horns vaginal smears were taken to confirm the presence of cornified epithelial cells. The smears were fixed with 100% methanol and were allowed to air dry before staining with Giemsa stain to aid microscopic visualization of cells. Timed pregnant rats (240 –390 g), at various stages of gestation, were housed as described above. They were killed as outlined above at either Days 17–19 or Days 20 –21 of the 22 day pregnancy. Fetuses were killed by cervical dislocation after their removal from the uterine horns. After vaginal smears were taken from estrogen-primed animals a cut was made through the abdominal muscle layers to expose the uterus. Both uterine horns were removed and placed on a Petri dish containing modified Krebs–Henseleit solution of the following composition: (mM: NaCl, 118.0; KCl, 4.7; CaCl2.2H2O, 1.9; MgSO4.7H2O, 1.1; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.7). Surrounding fat and connective tissue were removed, and each horn bisected transversely to produce four preparations from each animal (29). Myometrial segments from pregnant animals were obtained as outlined above. Placental
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and fetal membranes were removed, and segments of uterus from fetus-bearing horns were obtained. Uterine preparations were tied onto tissue holders and placed into jacketed, siliconized, isolated organ baths (5 or 10 ml) containing bathing solution warmed to 37°C. The bathing solution was continuously bubbled with 95% O2 in CO2 to maintain pH at 7.4. Each myometrial preparation was attached to a Grass FTO3 isometric force transducer connected to a MacLab recording instrument. All preparations were set up so that longitudinally arranged muscle fibers were orientated vertically. Tissues were allowed to equilibrate in modified Krebs–Henseleit solution for 30 min under an initial force of 1 g (non-gravid rats) or 2 g (gravid animals). The bathing solution was replaced three to four times during this period. Agonist Effects Discrete log concentration-response curves were obtained using one-half log-unit concentration increments. Preparations were exposed to each agonist concentration for 4 min, then washed with 2–3 replacements of the bath volume; 20 min elapsed before the next concentration was tested. Only one log concentration-response curve was obtained on each preparation. Each preparation was exposed to 40 mM KCl solution (mM: NaCl, 82.7; KCl, 40.0; CaCl2.2H20, 2.5; MgSO4.7H2O, 0.5; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.7) 20 – 40 min after the last concentration of peptide was washed from the baths to confirm tissue viability and the selectivity of antagonists and the enzyme inhibitor used. Endothelin Receptor Blockade Uterine preparations were exposed to the ETA receptor antagonist, BQ-123 (1 mM; Reference 16) or the ETB receptor antagonist, BQ-788 (1 mM; Reference 17) for 30 min following the initial equilibration period. Log concentration–response curves were then constructed to endothelin-1 or sarafotoxin S6b. The antagonists were replaced each time the bath was washed, and so remained in contact with the tissues throughout the experiment. Inhibition of Endopeptidase 24.11 SCH 39370 (10 mM) was used to inhibit endopeptidase 24.11 (36). It was added to the bath for 30 min after the initial equilibration period, and replaced each time the bath was washed during the construction of log concentrationresponse curves to endothelin-1, endothelin-2, endothelin-3, sarafotoxin S6b, and sarafotoxin S6c. In a separate series of experiments, the ability of SCH 39370 to inhibit responses to the endothelin-1 precursor, big endothelin-1, was investigated. Uterine preparations from estrogen-treated rats were exposed to carbachol (10 mM) twice, in order to obtain reproducible responses, using a similar protocol as that outlined above, except that tissues were exposed to agonist concentrations for 5 min. If the two
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FIG. 1. Representative traces showing contractions evoked by endothelin-1 (upper panel) and sarafotoxin S6c (lower panel) on myometrial preparations obtained from an estrogen-treated rat, and also to a bathing solution in which 40 mM KCl replaced 40 mM NaCl. Agonist addition is shown by arrows; W indicates a wash. Preparations were placed under an initial force of 1g. ET-1 evoked concentration-related contractions. Sarafotoxin S6c was inactive. Exposure to 40 mM KCl produced contractile responses of both tissues.
carbachol-evoked contractions differed substantially, a third application of carbachol was employed. After 15–20 min and extensive washing, a contractile response to either endothelin-1 (5 nM) or big endothelin-1 (12 nM) was obtained. After drug washout, SCH 39370 (10 mM) was added to some of the preparations, others acted as time controls. 30 min later, the contractile response to the original peptide was reassessed. Data Analysis Responses to agonists were measured as area under the force-time curve (g.s) over the 4-min period of agonist exposure and were normalized for tissue weight. Since in most instances the highest concentration of agonist that could be used did not produce maximal tissue responses it was not usually possible to calculate EC50 values for agonists. The relative potencies of the agonists used, however, and the effects of endothelin receptor antagonists and SCH 39370 on log concentration-response curves to agonists were examined using linear regression analysis of data points lying approximately between 15– 85% of the greatest response obtained, for estimation of potency ratios with 95% confidence limits. Fitted regressions were tested for linearity, parallelism and coincidence using procedures outlined in Documenta Geigy (8). The positions of pairs of lines meeting the criteria of linearity and parallelism were deemed to differ when the 95% confidence limits of potency ratios did not include one (26). The ratios of potency of the agonist in the absence and presence of an antagonist were then used to estimate the apparent pKB using the formula described by Furchgott (12) viz. log [antagonist concentration/potency ratio minus one]. Analyses of variance (followed by Tukey’s test) and Student’s paired or unpaired t tests were also used, p , 0.05 being the criterion for acceptance of statistical significance; ‘n’ refers to the number of animals.
Drugs Endothelin-1 (human, porcine) was obtained from Auspep or the American Peptide Company; endothelin-2 (human), endothelin-3 (human, rat), sarafotoxin S6b, sarafotoxin S6c and BQ-123 [cyclo(D-Asp-L-Pro-D-Val-L-Leu-D-Trp)] were obtained from American Peptide Company. Big endothelin-1 was obtained from Auspep. BQ-788 (Dmpc-g-MeLeu9-D-Trp(l-CO2CH3)-D-Nle-OH) was purchased from Novabiochem. Endothelin-1 (Auspep) and sarafotoxin S6b were made up in distilled H2O. The remaining peptides were first dissolved in 200 ml dimethyl sulphoxide (DMSO) before being made up to the required concentrations (10 or 100 mM for agonists; 1 mM for antagonists) in distilled H2O. The highest concentration of DMSO in the organ baths was 0.002%. SCH 39370 (N-[N-[1-(S)-carboxyl-3phenylpropyl]-(S)-phenylalanyl]-(S)-isoserine) was made up to a 1 mM stock solution in 0.1% NaHCO3. Myometrial activity was not influenced by either vehicle. Estradiol cypionate was dissolved in a small volume of 100% ethanol and then made up to 20 mg/ml in peanut oil; this solution was kept in the dark. RESULTS Tissues from Estrogen-primed Animals Agonist potency order . Endothelins -1, -2, and -3, and sarafotoxin S6b, produced concentration-dependent contractile responses of myometrium from estrogen-primed, non-pregnant rats. These were characterized by an increase in the frequency of spontaneous contractions arising from the longitudinally arranged smooth muscle layer at concentrations typically up to 1–3 nM, and a superimposed increase in basal force at higher concentrations. Figure 1 shows typical traces obtained in response to ET-1 and 40 mM KCl, whereas sarafotoxin S6c was ineffective. Endothelin-1 induces concentration-related uterotonic activity
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at concentrations of 3 nM; contractions are not maximal at 0.1 mM. In contrast, sarafotoxin S6b clearly produced maximal responses at this concentration (Fig. 2). The order of agonist potency was sarafotoxin S6b . endothelin-1 5 endothelin-2 . endothelin-3 . sarafotoxin S6c (Fig. 2). The mean log concentration-response curve to sarafotoxin S6b lay almost four-fold to the left of that to endothelin-1 (potency ratio 5 3.68; 95% confidence limits 5 1.51, 10.37; df 5 21) whereas endothelin-3 was 8.5 times less potent (potency ratio endothelin-1 to endothelin3 5 8.50; 95% confidence limits 5 4.22, 22.43; df 5 39). Sarafotoxin S6c lacked uterotonic activity in concentrations of up to 0.1 mM in all four preparations in which it was tested (Fig. 1 and 2).
FIG. 2. Graph showing mean log concentration–response curves constructed using preparations of uterus from non-pregnant rats to endothelin-1 (E), endothelin-2 (M), endothelin-3 (‚), sarafotoxin S6b (F) and sarafotoxin S6c (f). Vertical axis represents the area under the force-time curve, normalized for tissue weight. Responses are shown as mean, n 5 4 – 6; vertical bars represent SEM.
Effects of Endothelin Antagonists Responses to endothelin-1 and sarafotoxin S6b were significantly inhibited by the ETA receptor antagonist BQ-123 (1 mM; Figs. 3A & B). The magnitude of the rightward shifts of the concentration-response curves to these peptides in the presence of BQ-123 were 20.23-fold for endothelin-1 (95% confidence limits 5 11.24, 47.53; df 5 20) and 35.99-fold
FIG. 3. Log concentration–response curves of uterus from non-pregnant rats to endothelin-1 or sarafotoxin S6b in the absence (E) or presence of BQ-123 (1 mM; F; A and B, respectively) or BQ-788 (1 mM; F; C and D, respectively). Responses are shown as mean; vertical bars represent SEM; n 5 4 – 6. *p , 0.05, ***p , 0.001, compared to corresponding point of control curve (Tukey’s test). Peptide-evoked contractions were attenuated by BQ-123 but not by BQ-788.
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TABLE 1 POTENCY RATIOS OBTAINED FROM LINEAR REGRESSION ANALYSIS OF LOG CONCENTRATION-RESPONSE CURVES CONSTRUCTED IN TISSUES FROM NON-PREGNANT RATS IN THE ABSENCE AND PRESENCE OF SCH 39370 (10 mM) Peptide
Number of Tissues
Potency Ratio
95% Confidence Limits (df)
Endothelin-1 Endothelin-2 Endothelin-3 Sarafotoxin S6b Sarafotoxin S6c
6 6 5 5 3
1.33 1.56 1.31 1.16 *
0.55, 3.89 (38) 0.91, 2.77 (43) 0.77, 2.34 (31) 0.61, 2.23 (26) *
* Indicates not determined. In each case, confidence limits included 1.0, indicating no significant effect of SCH 39370.
for sarafotoxin S6b (95% confidence limits 5 15.98, 131.23; df 5 20). pKB values calculated from these shifts were of 7.23 and 7.54, respectively, for the two peptides. The ETB receptor antagonist, BQ-788 (1 mM) had no effect upon log concentration-response curves to endothelin-1 or sarafotoxin S6b (Figs. 3C & D). Effects of SCH 39370 Experiments were conducted concurrently with the experiments yielding the control curves shown in Fig. 2. The endopeptidase 24.11 inhibitor, SCH 39370 (10 mM), had no effect upon responses of myometrium to the peptides (Table 1). In a separate series of experiments, uterine preparations from six estrogen-treated rats were exposed to carbachol (10 mM). All preparations responded consistently (n 5 6; p . 0.05). The mean response was 7.08 6 0.45 g.s/mg tissue (n 5 24 preparations from 6 animals). The subsequent addition of ET-1 (5 nM) caused contractions of 4.53 6 1.07 g.s/mg (Fig. 4). Responses to big endothelin-1 (12 nM) were significantly lower (p , 0.05) than those obtained to endothelin-1, giving values of 1.88 6 0.53 g.s/mg tissue SCH 39370 was without effect on responses to ET-1, but significantly reduced those to big ET-1 (Fig. 4). Tissues from Pregnant Animals Agonist potency order . All preparations responded similarly to the modified KCl bathing solution. The contractures induced were evident immediately and were usually sustained for the duration of measurement. Previous exposure to a peptide agonist produced no difference in the magnitude of responses elicited by subsequent exposure to the high potassium solution. The relative orders of agonist potencies were similar at Days 17–19 and at Days 20 –21 (Fig. 5). Thus the potency order in tissues from both groups of animals was sarafotoxin S6b 5 ET-1$ ET-2 . ET-3. Sarafotoxin S6c was not investigated. Interestingly, the 1og concentration effect curves to sarafotoxin S6b in tissues
FIG. 4. Histograms showing the effects of ET-1 and big ET-1 on preparations of myometrium from estrogen-primed rats before and after exposure to SCH 39370 (10 mM). The vertical axis represents the area under the force-time curve, normalized for tissue weight. Each column represents the mean six experiments; vertical bars represent SEM. *p , 0.05, Student’s paired t-test.
from pregnant animals did not plateau at the highest concentrations used (Fig. 5) as they did in tissues from nonpregnant estrogen-primed animals (Fig. 2). The potency of this peptide was similar to rather than greater than that of ET-1, as was the case in tissues from the non-pregnant animals (Table 2). Furthermore, responses to the maximal concentrations of each peptide used, namely 0.1 mM, when normalized for tissue weight, were similar at the two stages of pregnancy (Fig. 5) and these responses were generally similar to those observed in tissues from non-pregnant animals (ANOVA, p . 0.05). Effects of SCH 39370 The effects of SCH 39370 (10 mM) on responses to the peptides in preparations from pregnant rats are shown in Figs. 6 and 7. Responses of myometrium from day 17–19 pregnant rats to endothelins-1 and -3 and to sarafotoxin S6b were not modified by SCH 39370, but there was some enhancement of the effects of endothelin-2 (Fig. 6). In this set of experiments, but in no others, there was also an enhancement by SCH 39370 of the effects of high potassium solution indicating that the effect on endothelin-2 was not selective. On myometrium from D20 –21 pregnant rats, the mean concentration-response curves of the four peptides were unaltered in the presence of SCH 39370 (Fig. 7). DISCUSSION The major, albeit unexpected, finding of this study was that the enzyme endopeptidase 24.11 does not limit the uterotonic effects of endothelins in uterine preparations from
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FIG. 5. Graphs showing mean log concentration–response curves to endothelin-1 (E), endothelin-2 (M), endothelin-3 (‚), and sarafotoxin S6b (F) in tissues taken from rats at Days 17–19 (D17–19) and Days 20 –21 (D20 –21) of pregnancy. Vertical axes represent the area under the force–time curve, normalized for tissue weight. Each point represents the mean of 4 – 6 experiments; vertical bars represent SEM.
estrogen-primed, non-pregnant rats or from late pregnant rats. The study also indicated that the endopeptidase 24.11 inhibitor used in this study, SCH 39370, may inhibit the conversion of big endothelin-1 to endothelin-1 in the uterus of the estrogen-primed, non-pregnant rat. The present study has also 1) confirmed that the endothelins and sarafotoxin S6b contract longitudinally-arranged smooth muscle of uterine horns from estrogen-primed rats and late pregnant rats by activating ETA receptors and 2) provided strong evidence that ETB receptors do not contribute to the actions of the peptides in tissues from the estro-
TABLE 2 POTENCY RATIOS OBTAINED FROM LINEAR REGRESSION ANALYSIS OF LOG CONCENTRATION RESPONSE CURVES CONSTRUCTED FOR ET-2, ET-3, AND SARAFOTOXIN S6B COMPARED TO CORRESPONDING CURVES FOR ET-1 IN PREPARATIONS FROM ESTROGEN PRIMED AND PREGNANT RATS Potency Ratio (95% CL; df)* Peptide Endothelin-2 Endothelin-3 Sarafotoxin S6b
Estrogen Treated
Pregnant: D17–19
Pregnant: D20–21
1.68 (0.88, 3.40; 44) 0.12† (0.05, 0.24; 39) 3.68† (1.51, 10.37; 21)
0.28† (0.07, 0.71; 28) 0.04† (0.02, 0.06; 12) 1.09 (0.61, 1.95; 25)
0.55 (0.19, 1.40; 33) 0.05† (0.02, 0.10; 24) 0.62 (0.17, 1.83; df)
* 95% confidence limits and degrees of freedom (df). † Indicates a significant difference in the location of the mean log concentrationresponse curves to that peptide relative to endothelin-1 (i.e., confidence limits do not include 1.0). A potency value greater than one indicates that the log concentrationresponse curve lay to the left of that for ET-1.
FIG. 6. Log concentration–response curves constructed using uterus taken from rats at Days 17–19 of pregnancy to ET-1, ET-2, ET-3, or sarafotoxin S6b in the absence (open symbols) or presence of SCH 39370 (10 mM; filled symbols). Responses are shown as mean of four experiments, vertical bars represent SEM. *p , 0.05 compared to corresponding point of control curve.
ENDOTHELINS AND EC 24.11 IN RAT UTERUS
FIG. 7. Log concentration–response curves constructed using uterus taken from rats at Days 20 –21 of pregnancy to ET-1, ET-2, ET-3, or sarafotoxin S6b in the absence (open symbols) or presence of SCH 39370 (10 mM; filled symbols). Responses are means of six experiments; vertical bars represent SEM.
gen-primed group. Our confirmation of earlier work (30,37) that ETA but not ETB receptors mediate endothelin-induced contractions of rat longitudinal myometrium of estrogenprimed, non-pregnant rats is based on findings that (1) endothelin-3 was less potent as a uterotonic agent than endothelins -1, -2, and sarafotoxin S6b; (2) sarafotoxin S6c, which selectively activates ETB receptors (40), was ineffective, (3) the ETA -selective antagonist, BQ-123 (16) produced apparently competitive inhibition of responses of myometrium to endothelin-1 and sarafotoxin S6b, and (4) responses to the latter peptides were unaltered by the presence of the ETB-selective antagonist, BQ-788 (17). The apparent pKB value we obtained for BQ-123 versus endothelin-1 (7.23) is similar to the pA2 value of this antagonist reported by Rae et al. (30) for rat uterus. Our findings are of additional interest given that the concentrations of estrogen we used to prime animals were some ten times lower than those used in the earlier experiments. It is conceivable that sarafotoxin S6b may activate binding sites distinct from or additional to those activated by endothelin-1. We found, however, that BQ-123 inhibited the responses to both peptides on rat longitudinally-oriented
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myometrium to a similar extent, indicating that in this tissue these peptides bind to the same sites. The apparent pKB values of BQ-123 as an antagonist of endothelin-1 and sarafotoxin S6b on rat myometrium were comparable with those estimated previously with tissues from non-pregnant rat uterus (37), and in human myometrium (13). The relative order of agonist potency of the endothelin isopeptides on days 17–19 and days 20 –21 indicated that the ETA receptor mediates the effects of their contractile effects on the pregnant uterus. The response to KCl and the responses obtained to the maximum concentrations of the peptides were apparently greater in tissues from pregnant rats, however this increased responsiveness paralleled the considerable uterine hypertrophy characteristic of pregnancy. Thus responses of tissues from gravid rats, when normalized for tissue weight, did not exceed those obtained in tissues from non-pregnant animals. Other authors have reported an increased potency of endothelin-1 on rat gravid uterine preparations (3,32). These authors, however, expressed their data as absolute force, as area under the force-time curve, or as a percentage of the response to KCl. All of these measures are influenced by uterine hypertrophy. Yallampalli and Garfield (43) reported an increased ability of endothelin-1 to contract myometrial strips obtained from delivering rats (D22 of gestation) compared to preparations obtained from near-term pregnant rats (D18). This suggests a possible role for endothelins in labour. The present experiments did not address this possibility. Endothelin degradation can be effected by neutral endopeptidase 24.11 (EC 24.11). This is a multisubstrate (14,28,22) membranous metalloprotease which cleaves endothelin-1 preferentially between Leu5-Ser6; this is then followed by cleavage at the carboxy-terminus tail (34,38). Amino acid substitutions at the corresponding positions on endothelins-2 and -3 confer resistance to EC 24.11 at this site but promote cleavage at the tail. In the rat, the activity of myometrial EC 24.11 increases markedly during gestation, declines slightly just prior to parturition, and continues to fall post-partum (28). Hence, modulation of the activity of EC 24.11 offers a potentially significant mechanism for regulating the in vivo concentrations of endothelins. In contrast to the endothelins, the sarafotoxins are relatively resistant to degradation by EC 24.11 (34). Thus these peptides are useful tools in investigations of the functional significance of EC 24.11-mediated endothelin degradation. In tissues from pregnant animals, the potency of sarafotoxin S6b relative to that of endothelin-1 was reduced rather than increased. We are unable to offer an explanation for this decrease in relative potency, but it indicates that there are no pregnancy-related decreases in the susceptibility of the endothelin peptides to degradation by endopeptidase 24.11. Generally, inhibition of this enzyme by SCH 39370 had negligible effect on the potencies of any of the peptides in tissues from either non-pregnant or pregnant animals. An
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exception was seen in tissues taken from rats on Days 17–19 of pregnancy in which SCH 39370 enhanced responses to some concentrations of ET-2. In this group of experiments it also enhanced responses to high potassium. It is unlikely that the concentration (10 mM) of SCH 39370 we used to inhibit this enzyme are too low, as at 3 mM, it produces maximal potentiation of the effects of neurokinin A (10). The latter peptide is a good substrate for EC 24.11 (14). The possibility exists that EC 24.11 may not be the main contributor to endothelin degradation in the uterus. Fagny et al. (9) have described the ability of soy bean tryptase inhibitor, an inhibitor of serine proteases, including cathepsin G, to inhibit the appearance of [125I]-endothelin-1 degradation products from activated polymorphonuclear neutrophils. In their experiments, an EC 24.11 inhibitor, phosphoramidon, was devoid of any such effect. An interesting possibility is that cathepsin G rather than EC 24.11 may be involved in endothelin degradation in the uterus. A novel finding of this study was that SCH 39370 decreased the response of the uterine preparations from non-pregnant rats to big ET-1. As already described, SCH 39370 did not modify the endothelin-1-evoked contraction. SCH 39370 has previously been reported to be a selective inhibitor of EC 24.11 (36). Other EC 24.11 inhibitors, such as phosphoramidon (11,22) and CGS 26303 (S)-2-biphe-
nyl-4-yl-1-(iH-tetrazol-5-yl)-ethylamino-methyl phosphonic acid (7) also function as ECE inhibitors (30). In the absence of biochemical data it is premature to postulate whether the effect of SCH 39370 on the response to big ET-1 occurs through the inhibition of ECE or of another enzyme involved in the conversion of big ETs to endothelins. However, it is likely that administration of SCH 39370 would limit rather than exacerbate the involvement of endogenous endothelins in uterine contractility. In conclusion, this study has confirmed that the endothelins are potent stimulators of uterine contractility of tissues from pregnant as well as from non-pregnant rats. In this species, in contrast to the human (13,27) and the guinea-pig (18) their uterotonic effects are mediated exclusively by ETA receptors in both pregnant and non-pregnant states. Their effects are not significantly influenced by inhibition of EC 24.11, suggesting that this endopeptidase is not a significant determinant of endothelin potency in this tissue in this species, even during pregnancy when the levels of this enzyme are reported to increase (28). ACKNOWLEDGEMENTS We are indebted to Dr. E. Sybertz, for a generous gift of SCH 39370.
REFERENCES 1. Ambar, I.; Kloog, Y.; Schvartz, I.; Hazum, E.; Sokolovsky, M. Competitive interaction between endothelin and sarafotoxin: binding and phosphoinositides hydrolysis in rat atria and brain. Biochem. Biophys. Res Commun. 158:195–201;1989. 2. Arai, H.; Hori S.; Aramori, I.; Ohkubo, H.; Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature (London). 348:730 –732; 1990. 3. Calixto, J. B.; Rae, G. A. Effects of endothelins, Bay K8644 and other oxytocics in non-pregnant and late pregnant rat isolated uterus. Eur. J. Pharmacol. 192:109 –116; 1991. 4. Cameron, I. T.; Bacon, C. R.; Collett, G. P.; Davenport, A. P. Endothelin expression in the uterus. J. Steroid. Biochem. Mol. Biol. 53:209 –214; 1995. 5. Chou, J.; Wang, Y. N.; Chang, D.; Chang, J. K.; Avila, C.; Romero, R. Big endothelin in plasma and amniotic fluid. J. Cardiovasc. Pharmacol. 17(Suppl. 7):S430 –S433; 1991. 6. Davenport, A. P.; Cameron, I. T.; Smith, S. K.; Brown, M. J. Binding sites for iodinated ET-1, ET-2 and ET-3 demonstrated on human uterine glandular epithelial cells by quantitative high-resolution autoradiography. J. Endocrinol. 129:149 –154; 1991. 7. De Lombaert, S.; Ghai, R. D.; Jeng, A. Y.; Trapani, A. J.; Webb, R. L. Pharmacological profile of a non-peptidic dual inhibitor of neutral endopeptidase 24.11 and endothelin converting enzyme. Biochem. Biophys. Res. Commun. 204:407– 412; 1994. 8. Geigy Scientific Tables, Vol 2, 8th edn. Introduction to statistics, statistical tables, mathematical formulae, ed Lerner. Ciba–Geigy Ltd Basle, Switzerland, 1982, pp 214. 9. Fagny, C.; Michel, A.; Nortier, J.; Deschodt–Lanckman, M. Enzymatic degradation of endothelin-1 by activated human polymorphonuclear neutrophils. Regul. Pept. 42:27–37; 1992.
10. Fisher, L.; Pennefather, J. N. Potencies of agonists acting at tachykinin receptors in the estrogen-primed rat uterus: effects of peptidase inhibitors. Eur. J. Pharmacol., 335:221–226; 1997. 11. Fukuroda, T.; Noguchi, K.; Tsuchida, S.; Nishikibe, M; Ikemoto, F.; Okada, K.; Yano, M. Inhibition of biological actions of big endothelin-1 by phosphoramidon. Biochem. Biophys. Res. Commun. 172:390 –395; 1990. 12. Furchgott, R. F. The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory. In: Blaschko, H.; Muscholl, E., eds. Handbook of Experimental Pharmacology. Springer–Verlag: Berlin, vol. 33; 283–336; 1972. 13. He´luy, V.; Germain, G.; Fournier, T.; Ferre´, F.; Breuiller– Fouche´, M. Endothelin ETA receptors mediate human uterine smooth muscle contraction. Eur. J. Pharmacol. 285:89 –94; 1995. 14. Hooper, N. M.; Kenny, J; Turner, A. J. The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase (angiotensin-converting enzyme). Biochem. J. 231:357–361; 1985. 15. Huggins, J. P.; Pelton, J. T.; Miller, R. C. The structure and specificity of endothelin receptors: their importance in physiology and medicine. Pharmacol. Ther. 59, 55–123; 1993. 16. Ihara, M.; Noguchi, K.; Saeki, T.; Fukuroda, T.; Tsuchida, S.; Kimura, S.; Fukami, T.; Ishikawa, K.; Nishikibe, M.; Yano, M. Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor. Life Sci. 50:247–255; 1992. 17. Ishikawa, K.; Ihara, M.; Noguchi, K.; Mase, T.; Mino, N.; Saiki, T.; Fukuroda, T.; Fukami, T.; Ozaki, S.; Nagase, T.; Nishikibe, M.; Yano, M. Biochemical and pharmacological
ENDOTHELINS AND EC 24.11 IN RAT UTERUS
18.
19.
20.
22.
23. 24.
25.
26. 27.
28. 29. 30.
31.
profile of a potent and selective endothelinB-receptor antagonist, BQ-788. Proc. Natl. Acad. Sci. USA. 91:4892– 4896; 1994. Kousides, M.; Story, M. E.; Pennefather, J. N. Endothelin receptors mediating contractile response of rat and guinea-pig uterus. Proc. Aust. Soc. Clin. Exp. Pharmacol. Toxicol. 2, 51; 1995. Kozuka, M.; Ito, T.; Hirose, S.; Eguchi, S.; Hagiwara, H. Endothelin action on rat uterus is inhibited by an inhibitor of protein kinase C and by inhibitors of the phospholipase A2arachidonic acid-lipoxygenase pathway. Biomed. Res. 11:287–289; 1990. Kozuka, M.; Ito, T.; Hirose, S.; Takahashi, K.; Hagiwara, H. Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channel. Biochem. Biophys. Res. Commun. 159, 317–323; 1989. Malfroy, B.; Schwartz, J.C. Properties of ‘enkephalinase’ from rat kidney: comparison of dipeptidyl-carboxypeptidase and endopeptidase activities. Biochem. Biophys. Res. Commun. 106:276 –285; 1982. Masaki, T.; Vane J. R.; Vanhoutte, P. M. International union of pharmacology nomenclature of endothelin receptors. Pharmacol. Rev. 46, 137–142;1994. Nisell, H.; Hemse´n, A.; Lunell, N. O.; Wolff, K.; Lundberg, M. J. Maternal and fetal levels of a novel polypeptide, endothelin: evidence for release during pregnancy and delivery. Gynecol. Obstet. Invest. 30:129 –132; 1990. Ohbuchi, H.; Nagai, K.; Yamaguchi, M.; Ikenoue, T.; Mori, N.; Kitamura, K.; Araki, S.; Toshimori, K. Endothelin-1 and big endothelin-1 increase in human endometrium during menstruation. Am. J. Obstet. Gynecol. 173:1483–1490; 1995. Orre, M.; Pennefather, J. N.; Story, M. E.; Haynes, J. M. The effects of P2 purinoceptor agonists on the isolated portal vein of the guinea pig. Eur. J. Pharmacol. 316:229 –236; 1996. Osada, K.; Tsunoda, H.; Miyauchi, Y.; Sugishita, Y.; Kubo, T.; Goto, K. Pregnancy increases ET-1-induced contraction and changes receptor subtypes in uterine smooth muscle in humans Am. J. Physiol. 272:R541–548; 1997. Ottlecz, A.; Walker, S.; Conrad M.; Starcher, B. Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus, J. Cell. Biochem. 45:401– 411; 1991. Pennefather, J. N.; Zeng, X. -P.; Gould, D.; Hall, S.; Burcher, E. Mammalian tachykinins stimulate rat uterus by activating NK-2 receptors. Peptides. 14:169 –174; 1993. Rae, G. A.; Calixto, J. B.; D’Orle´ans–Juste, P. Big-endothelin-1 contracts rat isolated uterus via a phosphoramidon-sensitive endothelin ETA receptor-mediated mechanism. Eur. J. Pharmacol. 240:113–119; 1993. Riley, S. C.; Butt, A. R.; Doughton, B. W.; Li, S. Y.; Zheng,
1593
32. 33.
34.
35.
36.
37. 38.
39.
40.
41.
42. 43.
S. H.; Findlay, J. K.; Salamonsen, L. A. Endothelin in the ovine uterus during the oestrous cycle and early pregnancy. J. Reprod. Fertil. 100:451– 459; 1994. Sakata, K.; Karaki, H. Effects of endothelin on cytosolic Ca21 level and mechanical activity in rat uterine smooth muscle. Eur. J. Pharmacol. 221:9 –15; 1992. Sakurai, T; Yanagisawa, M; Takuwa, Y; Miyazaki, H.; Kinura, S.; Goto, K.; Masaki, T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of endothelin receptor. Nature (London). 348, 732–735; 1990. Sokolovsky, M.; Galron, R.; Kloog, Y.; Bdolah, A.; Indig, F. E.; Blumberg, S.; Fleminger, G. Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance. Proc. Natl. Acad. Sci. USA. 87, 4702– 4706; 1990. Svane D.; Larsson, B.; Alm, P.; Andersson, K. E.; Forman, A. Endothelin-1: immunocytochemistry, localization of binding sites, and contractile effects in human uteroplacental smooth muscle. Am. J. Obstet. Gynecol. 168, 233–241; 1993. Sybertz, E. J.; Chiu, P. J. S.; Vemulapalli, S.; Pitts, B.; Foster, C. J.; Watkins, R. W.; Barnett, A.; Haslanger, M. F. SCH 39370, a neutral metalloendopeptidase inhibitor, potentiates biological responses to atrial natriuretic factor and lowers blood pressure in desoxycortisone acetate-sodium hypertensive rats. J. Pharmacol. Exp. Ther. 250:624 – 631; 1989. Tsunoda, H.; Miyauchi, T.; Fujita, K.; Kubo, T.; Goto, K. Mechanism of rat uterine smooth muscle contraction induced by endothelin-1. Br. J. Pharmacol. 110:1437–1446; 1993. Vijayaraghavan, J.; Scicli, A. G.; Carretero, O. A.; Slaughter, C.; Moomaw, C.; Hersh, L. B. The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase). J. Biol. Chem. 265:14150 –14155; 1990. Williams, D. L.; Jones, K. L.; Pettibone, D. J.; Lis, E. V.; Clineschmidt, B. V. Sarafotoxin S6c, an agonist which distinguishes between endothelin receptor subtypes. Biochem. Biophys. Res. Commun. 175, 556 –561; 1991b. Williams, D. L.; Jones, K. L.; Colton, C. D.; Nutt, R. F. Identification of high affinity endothelin-1 receptor subtypes in human tissues. Biochem. Biophys. Res. Commun. 180:475– 480; 1991a. Wollberg, Z.; Bousso–Mittler, D.; Bdolah, A.; Kloog, Y.; Kochva, E.; Sokolovsky, M. Endothelins and sarafotoxins: effects on motility, binding properties and phosphoinositide hydrolysis during the estrous cycle of the rat uterus. J. Basic Clin. Physiol. Pharmacol. 3:41–57; 1992a. Wollberg, Z.; Shinnar, N.; Bdolah, A.; Kochva, E. Endothelin and sarafotoxin: influence on steroid-regulated motility of rat uterus. Life Sci. 51, L57– 60; 1992b. Yallampalli, C.; Garfield, R. E. Uterine contractile responses to endothelin-1 and endothelin receptors are elevated during labor. Biol. Reprod. 51:640 – 645; 1994.