European Journal of Pharmacology, 192 (1991) 109-116 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0014-2999/91/$03.50 ADONIS 001429999100075T
109
EJP 51623
Effects of endothelins, Bay K 8644 and other oxytocics in non-pregnant and late pregnant rat isolated uterus Jo~o B. Calixto a n d Giles A. R a e Department of Pharmacology (CCB), Universidade Federal de Santa Catarina, 88049 Florian@olis, SC, Brazil Received 11 July 1990, accepted 25 September 1990
Endothelins 1, 2 and 3 (ET-1, ET-2 and ET-3; 1-30 nM) caused long-lasting concentration-dependent tonic contractions of uterine strips from non-pregnant rats. The potency of ET-1 (ECs0 7 nM) was similar to that of angiotensin II (AII) and greater than that of ET-2 or ET-3 (ECs0s >i 10 nM), bradykinin, Bay K 8644, oxytocin (OT), 5-hydroxytryptamine, prostaglandin F2, (PGF2~) or acetylcholine. Strips from 21-day pregnant rats were 2- to 3-fold more sensitive to ET-1, AII, OT and PGF2~ and 200-fold more sensitive to Bay K 8644 than non-pregnant preparations. The development of tonic responses to ET-1 (30 nM) and of phasic-rhythmic ones to Bay K 8644 (300 nM) was fully prevented in strips from non-pregnant rats bathed in Ca2+-free medium, but stepwise reintroduction of Ca :+ (0.03-3 mM) to the solution allowed the manifestation of contractions in response to both agonists. Responses to ET-1 required less Ca 2÷ than those to Bay K 8644. Strips challenged with ET-1 while in Ca2+-free medium developed greater contractions upon reintroduction of Ca 2+ than preparations stimulated with the peptide in normal medium. The reverse occurred with Bay K 8644-induced contractions. Nicardipine (10 nM) abolished the responses of strips from non-pregnant rats to Bay K 8644 (300 nM), but only attenuated ET-l-induced (30 nM) contraction. A subthreshold concentration of ET-1 (0.3 nM) augmented the responses to PGF2~ or 5-HT and to AII in preparations taken from non-pregnant and pregnant rats. Thus, ET-1 is one of the most powerful known constrictors of the rat isolated uterus. Its action is enhanced during late pregnancy and is mediated by a mechanism distinct from that of Bay K 8644. It is suggested that ETs may play significant roles in the control of uterine contractility. Endothelins; BAY k 8644; Oxytocic agents; Uterus (rat); Nicardipine; Pregnancy
1. Introduction
Recently, Yanagisawa et al. (1988) demonstrated that cultured porcine aortic endothelial cells release endothelin (ET) a 21-residue peptide of unusual structure with potent and long-lasting vasoconstrictor and pressor activities in vitro and in vivo, respectively. On a molar basis, the constrictor potency of ET on coronary artery rings isolated from pigs was found to be 100-fold greater than that of the potent vasoconstrictor, angiotensin II. It has since become evident (Inoue et al., 1989) that the human, rat and porcine genomes each contain three distinct isogenes encoding different ET-like peptides: ET-1 (previously named porcine ET), ET-2 and ET-3 (previously called rat ET). Also, these peptides show striking structural and pharmacological resemblance with the sarafotoxins present in the Atractaspis engaddensis snake venom (Kloog and Sokolovsky, 1989).
Correspondence to: J.B. Calixto, Department of Pharmacology, CCB, Universidade Federal de Santa Catarina, Ferreira Lima 26, 88015 Florian6polis, SC, Brasil.
Besides its potent vasoconstrictor effects (Yanagisawa et al., 1988; De Nucci et al., 1988), ET-1 causes positive inotropic and chronotropic effects on the heart (Hu et al., 1989; Ishikawa et al., 1988; Moravec et al., 1989), stimulates cell proliferation (Komuro et al., 1988; Simonson et al., 1989), triggers the release of eicosanoids (Rae et al., 1989) and atrial natriuretic peptide (Fukuda et al., 1988), inhibits renin release (Rakugi et al., 1988) and affects sympathetic neurotransmission (Wiklund et al., 1988; Tabuchi et al., 1989). Also, in vitro, ET-1 contracts non-vascular smooth muscle such as that of the rat stomach (De Nucci et al., 1988), guinea-pig trachea (Maggi et al., 1989a) human urinary bladder (Maggi et al., 1989b) and bronchi (Uchida et al., 1988). A recent study has also shown the marked oxytocic activity of ET-1 on rat uterine strips (Kosuka et al., 1989). Since the contractility of uterine smooth muscle is highly susceptible to modulation by sex hormones (Fuchs, 1978; Riemer and Roberts, 1986), the present study compared the effects of ET-1, ET-2 and ET-3 on the uterus isolated from non-pregnant and late pregnant rats with the effects of several known oxytocic agents. Also, because it was proposed that ET-1 is a putative
110 endogenous agonist of L-type voltage-dependent (dihydropyridine-sensitive) Ca 2+ channels (Yanagisawa et al., 1988) and was shown to strongly activate such channels (Goto et al., 1989; Silberberg et al., 1989), we compared its profile of activity with that of the dihydropyridine agonist, Bay K 8644. Finally, we assessed the influence of ET-1 on contractions induced by other oxytocic agents.
2. Materials and methods
2.1. Animals Adult female Wistar rats (180-250 g) kept in a room with controlled temperature (22 + 2°C) and illumination (12 h light and 12 h darkness) were used. Nonpregnant virgin animals were pretreated with oestradiol benzoate (0.5 mg/kg s.c.) 24 h before the experiments. Pregnant animals were obtained by pairing groups of 10 virgin female rats with three males overnight only. The rats that became pregnant were separated and killed 21 days later.
2.2. Experimental procedures The animals were killed by a blow on the head and their uteri were removed and carefully dissected free from adhering tissue. Pregnant uteri were cut transversally at points between foetus implants and the foetuses and placentas were gently removed. Uterine strips of 15 mm in length were suspended in 5-ml jacketed organ baths at 30 °C bubbled with air, containing physiological De Jalon solution of the following composition (mM): NaC1 154; KC1 5.6; CaCI 2 0.3; MgC12 1.4; NaHCO 3 1.7; and glucose 5.5. Isotonic contractions were measured under a resting load of 1 g using a light lever (six-fold amplification) writing on a kymograph. The tissues were equilibrated for at least 30-45 min before drug additions, during which time the bath solution was renewed every 15 min. After this period the preparations were first exposed to KC1 (80 mM)-rich solution (by equimolar replacement of 80 mM of NaC1 by KC1 in the medium). After washout, replacement with normal solution and complete relaxation (20-30 min), concentration-response curves were obtained to one of several agonists. Endothelins (ET-1, ET-2 and ET-3), Bay K 8644, bradykinin (BK), acetylcholine (ACh) and oxytocin (OT) were added to the bath in stepwise cumulative concentrations. Curves to 5-hydroxytryptamine (5-HT), angiotensin II (AII) and prostaglandin F2~ (PGF2~) were obtained by adding single increasing concentrations of the agonist to the bath for 30-60 s at 10-15 min intervals. The potencies of the various agonists were evaluated at the ECs0 level, i.e. at concentrations producing 50% of the maximal response.
Another set of experiments served to determine the influence of external Ca 2÷ on contractile responses of strips from non-pregnant rats to ET-1 (30 nM) and Bay K 8644 (300 nM). Following equilibration, the preparations were contracted with KC1 (80 mM) as before. The preparations were then bathed in Ca2+-free De Jalon solution containing 0.2 mM EGTA for 20 min, during which the solution was renewed every 5 min. This was followed by another 10 min in Ca2÷-free solution (without EGTA) before the addition of ET-1 (30 nM) or Bay K 8644 (300 nM) to the bath. After confirming the absence of a contractile response in Ca2÷-free solution and still in presence of the agonist, Ca 2÷ (0.03-3 mM) was added cumulatively to the bath. The contractions obtained in this way, i.e. by adding the agonist in Ca2+-free solution then reintroducing Ca 2+ to the bathing medium, were compared to those elicited by the agonist in time-matched strips taken from the same animals and bathed in normal solution (Ca 2÷ 0.3 mM) throughout the experiment. To analyse the possible participation of dihydropyridine-sensitive Ca 2÷ channels in the start of contractions induced by ET-1, uterine strips from non-pregnant rats were exposed to a single concentration of nicardipine (10 or 100 nM) and challenged 20 min later with ET-1 (30 nM) or Bay K 8644 (300 nM). The responses to either agonist obtained in the presence of the dihydropyridine antagonist were compared to those caused by ET-1 or Bay K 8644 alone in different, time-matched, strips from the same animals. We also evaluated the influence of nicardipine on established contractions induced by ET-1 (30 nM) or Bay K 8644 (300 nM). In these experiments cumulative concentrations of the Ca2÷-channel antagonist (10-100 nM) were added to the medium once responses to the agonists had stabilized and the degree of inhibition was measured 20 min after each addition. The influence of ET-1 on the contractile responses of strips from non-pregnant and late pregnant rats to other agonists was assessed in a separate sequence of experiments. After a response to KC1 (80 nM) had been obtained, the preparations were stimulated repeatedly at 10- to 15-min intervals with a submaximally effective concentration (EC40 to EC70) of a given agonist. Once the contractions became reproducible, ET-1 (0.3 mM) was added to the bath for 10 rain and a new contraction in response to the agonist was obtained in its presence. In all experiments the contraction induced by KC1 (80 mM) was taken as the 100% response and the effects of the various agonists are presented as functions of this value.
2.3. Statistics The ECs0s are presented as geometric means accompanied by their 95% confidence limits. All other values
111
are presented as means + S.E.M. Statistical analyses were performed by means of two-tailed paired and unpaired Student's t-tests where appropriate, and P < 0.05 was considered significant.
3. Results
3.1. Effects of endothelins and Bay K 8644 on uterine strips from non-pregnant and pregnant rats
2.4. Drugs The drugs used were: ET-1 (porcine), ET-2 (human) and ET-3 (rat; all from the Peptide Institute Inc., Japan), Bay K 8644 (methyl-l,4-dihydro-2,6-dimethyl-3-nitro-4(2-tri-fluoromethylphenyl)-pyridine-5-carboxylate from Bayer A.G., FRG), bradykinin, acetylcholine iodide, 5-hydroxytryptamine creatinine sulphate, nicardipine, oestradiol benzoate (all from Sigma Chemical Company, USA), prostaglandin F2~ (Chinoin, Hungary), oxytocin (Syntocinon~, Sandoz, Brazil) and angiotensin II (synthesized by the Department of Biophysics, Escola Paulista de Medicina, $5o Paulo, Brazil). Most drugs were stored as 1-100 mM stock solutions at - 2 0 ° C and diluted to the desired concentration in PBS solution just before use. Endothelins were diluted in PBS solution from 10/~M stock solutions stored at - 2 0 ° C. The stock solutions of Bay K 8644 and nicardipine (10 raM) were made up in absolute ethanol and all others were prepared in PBS solution. Oestradiol was dissolved in peanut oil to 1 mg/ml. Solutions containing Bay K 8644 or nicardipine were protected from light and the experiments were carried out in the dark with these compounds to avoid photodegradation.
Cumulative additions of ET-1, ET-2 and ET-3 (0.1-30 nM) caused concentration-dependent tonic contractions of both non-pregnant and pregnant rat uterine strips, which were only slowly reversible following washout. Contractions induced by the highest concentration of ET-1 or ET-2 (30 nM) failed to subside completely within 60 min of washout, despite solution renewal every 10-15 min. The mean results depicted in fig. 1A-C and summarized in table 1 show that ET-1 was more potent than ET-2 and ET-3 to cause contractions of non-pregnant uterine strips. Although complete concentration-response curves for the ETs were not obtained due to limited availability of the peptides, contractions caused by the highest ET-1 concentration tested (30 nM) were comparable to those elicited by KC1 (80 mM) and significantly greater than responses to ET-2 or ET-3 (30 nM). The contractile concentration-response curve to ET-1 with 21-day pregnant rat uterine strips was shifted to the left about 2-fold relative to that obtained in preparations from non-pregnant animals (P < 0.05), as evaluated at the apparent ECs0 level (fig. 1A and table 1). ET-2 and ET-3 also seemed to be more potent in
TABLE 1 Responsiveness of non-pregnant and late pregnant (21 days) rat isolated uterus to endothelins 1, 2 and 3, Bay K 8644 and other oxytocic agents. Agonist
Endothelin-I Endothelin-2 Endothelin-3 Bay K 8644 Angiotensin II Bradykinin Serotonin Prostaglandin F2,~ Acetylcholine Oxytocin
Non-pregnant
Late pregnant
ECs0"
Maximal b response
ECs0
6.9 nM (5.3-8.8) >~10 nM >~10 nM 21.2 nM (14.5-39.1) 8.8 nM (6.7-11.6) 16.5 nM (10.4-26.2) 0.3/zM (0.1-0.5) 1.0/~M (0.7-1.5) 1.0 #M (0.7-1.4) 50.0 nM (37.1-62.5)
102.5_+ 7.5 c
3.8 nM d (2.7-5.2) >i 10 nM t> 10 nM 0.1 nM d (0.04-0.3) 2.6 nM a (1.8-3.8) 6.5 nM d (1.9-21.7) 0.4 ~M (0.2-0.6) 0.3/.tM d (0.2-0.7) 1.0 ~M (0.7-1.5) 16.7 nM d (12.5-25.0)
70.3-+ 5.4 c 21.9 + 12.8 ¢ 119.4+ 5.8 99.7+ 2.0 122.1-+ 5.5 116.7_+ 6.6 116.1 + 5.3 118.5 + 4.5 110.1 + 7.2
Maximal response 86.4-1- 5.9 c 62.45- 5.8 c 35.4 + 9.2 c 106.2+ 3.4 d 199.2+ 9.5 66.2+ 8.3 d 90.9+ 9.2 d 100.0+ 11.1 141.9 + 11.0 d 118.0+ 7.5
" ECs0s presented as the geometric means accompanied by 95% confidence limits, b Calculated as percentages of the response to KCI 80 mM (mean-+ S.E.M. of five to eight observations), c Response to highest concentration tested (30 nM). d p < 0.05 when compared to non-pregnant group.
112 i20'
Bay K 8644 (1-1000 nM) caused concentration-dependent phasic contractions of the non-pregnant rat uterus which were relatively resistant to washout. The pattern of contractions induced by Bay K 8644 was markedly different from that obtained with ET-1 (compare figs. 4A and C). Also in contrast to endothelin-induced contractions, the responses to Bay K 8644 were markedly influenced by pregnancy, as evidenced by an about 200-fold shift of the concentration-response curve at the ECs0 level and a slight but significant reduction of the maximal response (fig. 1D and table 1). Thus, Bay K 8644 was about 3-fold less potent than ET-1 in preparations from non-pregnant rats, but nearly 40-fold more potent than the peptide in strips from late pregnant animals.
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3.2. Contractions of the non-pregnant and late pregnant rat uterus induced by other oxytocics
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0t O.i i 10 tO0 1000 ENDOTHELIN-5 (nM) BAY K 8 6 4 4 (n M} Fig. 1. Mean contractile concentration-response curves for endothelin1 (A), endothelin-2 (B), endothelin-3 (C) and for Bay K 8644 (D) in the isolated uterus from 21-day pregnant (e) and non-pregnant (o) rats. Each point represents the mean of five to eight experiments from at least two different animals and the vertical bars indicate the S.E.M.
The mean contractile concentration-response curves for AII, BK, 5-HT, PGF2~, ACh and OT are illustrated in fig. 2. All the agonists caused concentration-dependent contractions of the non-pregnant uterus with the following rank order of potency: A I I > BK > OT > 5HT > PGF2~ = ACh (table 1). In preparations isolated from late pregnant rats, the contractile concentrationresponse curves for AII, PGF2~ and OT were significantly shifted to the left about 3-fold (P < 0.05), while the sensitivities to BK, ACh and 5-HT did not differ
pregnant strips, but an accurate estimation of this phenomenon was again hampered by the impossibility of making complete curves (fig. 1B, C and table 1). t40"
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Fig. 2. Mean contractile concentration-response curves for angiotensin II (A), bradykinin (B), serotonin (C), prostaglandin F2~ (D), acetylcholine (E) and oxytocin (F) in the uterus isolated from 21-day pregnant (e) and non-pregnant (0) rats. Each point represents the mean of 6 to 10 experiments with preparations from at least two different animals and the vertical bars indicate the S.E.M.
113 f r o m those of strips from n o n - p r e g n a n t rats (table 1), yielding the rank order of potency: A I I > B K > O T > 5 - H T = PGF2~ > ACh. Maximal contractile responses to the various agonists were also differentially affected by pregnancy (table 1). Whereas the maximal contractions induced by A I I and A C h were enhanced significantly, those induced b y BK and 5 - H T were reduced (P < 0.05). The maximal responses to PGFz~ and O T were similar in tissues from pregnant and n o n - p r e g n a n t rats.
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3.3. Influence of extracellular calcium content and nicardipine on effects of endothelin-1 and Bay K 8644 C"
Uterine preparations from non-pregnant rats, after equilibration for 30 min in Ca2+-free m e d i u m that contained 0.2 m M E G T A for the first 20 min, were completely refractory to the contractile effects of ET-1 (30 nM) and Bay K 8644 (300 nM). However, subsequent stepwise reintroduction of Ca 2+ (0.01 to 3 m M ) to the bathing solution, in the presence of either substance, p r o m p t l y restored their contractile effects in a concentration-related m a n n e r (fig. 3). In such experiments, the tonic response to ET-1 (fig. 3B) was about 3-fold more sensitive to Ca z+ reintroduction, with ECs0 0.20 m M (0.12-0.30, n = 5), than the phasic-rhythmic response to Bay K 8644, ECs0 0.63 m M (0.47-8.5, n = 7) (fig. 3D). Also, the strips challenged with ET-1 in CaZ+-free m e d i u m then exposed to increasing extracellular Ca 2+ concentrations (0.3-3 raM) showed significantly greater responses than time-matched preparations stimulated with the peptide while in normal D e Jalon solution (0.3 m M Ca 2+) (compare figs. 3A and B). In contrast, preparations challenged with Bay K 8644 in Ca2+-free solution exhibited contractions u p o n Ca 2+ reintroduction (fig. 3D) which were significantly
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CoCI2 CONCENTRATION (raM) Fig. 4. Influence of extracellular calcium concentration on the magnitude of contractions induced by endothelin-1 (30 nM) and Bay K 8644 (300 nM) in the non-pregnant rat isolated uterus. The preparations were exposed to the agonists either in Ca2+-free medium (open columns) or in normal De Jalon solution containing 0.3 mM Ca 2÷ (hatched columns). Responses were recorded in presence of the extracellular Ca 2+ concentrations indicated in the figure. Each column represents the mean of five to eight experiments and the vertical bars indicate the S.E.M. The responses to either agonist, added in the presence or absence of extracellular Ca 2÷, were significantly different at any given concentration of Ca 2+ (P < 0.05).
smaller than those obtained in strips bathed in normal De Jalon solution at the time of agonist challenge (fig. 3C). The means for the results of these experiments are presented in fig. 4. It is also noteworthy that, in the records illustrated in figs. 3C, D, with concentrations of Ca 2+ in excess of 0.3 mM, the responses to Bay K 8644 exhibited an additional tonic component. Preincubation of strips f r o m n o n - p r e g n a n t rats with nicardipine (10-
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Fig. 3. Representative isotonic tracings for the influence of extracellular calcium concentration on contractions induced by endothelin-1 (ET-1, 30 nM) or Bay K 8644 (300 nM) in non-pregnant rat isolated uterus. Note that in the experiments of panels A and C the agonists were added to strips bathed in normal De Jalon solution containing 0.3 mM Ca 2÷. In the experiments of panels B and C the preparations were equilibrated for 30 min with Ca2+-free solution (plus EGTA 0.2 mM for the first 20 min) and challenged with the agonist in CaZ+-free medium. Cumulatively increased concentrations of Ca 2+ were added to the bath as indicated in the figure. Similar results were obtained in another four experiments.
114 NON-PREGNANT
iO0" Z
pregnant rats to BK interfered with the analysis of the influence of ET-1 on BK-induced responses. Also, we were unable to analyse possible interactions between ET-1 and Bay K 8644 in strips from either non-pregnant or pregnant rats, because of the very slow reversibility of contractions caused by the dihydropyridine.
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Fig. 5. Influence of prcincubation with endothe]Jn-] (0.3 n]Vl) on the
contractile response induced by angiotensin II (AII), serotonin (5-HT), prostaglandin F2~ (PGF2~), acetylcholine (ACh), oxytocin (OT) and bradykinin (BK) in the non-pregnant (A) and 21-day pregnant (B) rat isolated uterus. Open columns represent control responses and hatched columns indicate the responses obtained in the presence of endothelin-1. Each column represents the mean of five to eight experiments and the vertical lines indicate the S.E.M. Endothelin-1 significantly enhanced the responses of strips from non-pregnant rats to 5-HT and PGF2~ and of 21-day pregnant strips to All (P < 0.05).
100 nM) for 20 rain caused partial graded inhibition of the development of responses to ET-1 (30 nM). At 10 and 100 nM, nicardipine reduced the height of contractions caused by ET-1 from 126.8 + 5.6% of the response to KC1 80 mM to 51.7 + 7.4 and 39.2 + 10.4%, respectively (n = 5). Established contractions induced by ET-1 were somewhat less susceptible to inhibition, so that about half of the original control response persisted in the presence of 100 nM of the dihydropyridine antagonist (59.8 + 7.5% of the response to KC1 80 mM, n = 5). In contrast, nicardipine at 10 nM fully abolished both the start and maintenance of Bay K 8644-induced responses (300 nM, n = 6). 3.4. Influence of endothelin-1 on contractions induced by
other oxytocics Figure 5A shows that incubation of non-pregnant rat uterine strips for 10 rain with a low concentration of ET-1 (0.3 nM), which alone did not cause contraction, significantly potentiated the responses to submaximally effective concentrations of 5-HT (10-100 nM) or PGF2~ (1/~M). However, the presence of ET-1 (0.3 nM) failed to alter contractions elicited by All (3 nM), BK (3 nM), ACh (1/xM) or OT (120 nM). In preparations from late pregnant rats, ET-1 potentiated only the responses to All (fig. 5B). The marked tachyphylaxis of strips from
4. Discussion
The results of the present study demonstrated that ET-1, ET-2 and ET-3 cause longqasting and concentration-dependent tonic contractions of uterus isolated from the non-pregnant rat. The rank order of potency was ET-1 > ET-2 >~ ET-3. On a molar basis, the potency of ET-1 was similar to that of All and greater than that of BK, Bay K 8644, OT, 5-HT, PGF2~ and ACh. Preparations isolated from rats on the 21st day of gestation were 2- to 3-fold more sensitive to ET-1, AII, OT and PGF2~ than strips from non-pregnant animals, whereas the sensitivity to Bay K 8644 was increased 200-fold. These results indicate that ET-1 is a remarkably potent oxytocic agent and that its action, like that of several (but not all) other agonists in this tissue, is susceptible to hormonal modulation. The markedly different patterns of contraction induced by ET-1 and Bay K 8644 in non-pregnant rat uterine strips further substantiate the view that ETs do not act as initially proposed (Yanagisawa et al., 1988) by direct activation of voltage-dependent L-type Ca 2+ channels. Bay K 8644, a dihydropyridine which activates such channels directly, caused phasic-rhythmic contractions, similar to those caused by OT. The rhythmicity of these responses may have been determined by intermittent or recurrent opening of high conductance C a 2+activated potassium channels (Watson and Abbott, 1990), leading to transient reductions of the ability of Bay K 8644 to activate L-type Ca 2+ channels (Triggle and Rampe, 1989). In contrast, ET-1 caused typically tonic contractions which, unlike those triggered by Bay K 8644, were only partially blocked by high concentrations of nicardipine. The finding that both ET-1 and Bay K 8644 failed to contract preparations bathed in CaE+-free solution indicates that responses to either agonist depended critically on the influx of Ca 2+ from the extracellular medium. Therefore, as previously demonstrated with vascular smooth muscle (Van Renterghem et al., 1988), it appears that uterine contractions induced by ET-1 involved primarily the influx of Ca 2+ through nicardipine-insensitive non-selective cation channels. The ensuing depolarization would then lead to activation of voltage-dependent L-type C a 2+ channels, accounting for the nicardipine-sensitive component of the ET-1 action. Taken together, the current data are consistent with reports showing that ET-1 fails to displace radio-labelled dihydropyridine antagonists
115 bound to smooth muscle membranes (Gu et al., 1989b), that it activates L-type Ca 2÷ channels indirectly (Silberberg et al., 1989) by opening non-selective cation channels (Van Renterghem et al., 1988) and causes contractions which are distinct from those induced by Bay K 8644 in isolated blood vessels (Auguet et al., 1988; D'Odrans-Juste et al., 1989). In vascular smooth muscle ET-1 stimulates phosphoinositide metabolism and mobilizes intracellular Ca 2÷ stores (Van Renterghem et al., 1988), possibly by raising the cytoplasmic levels of inositol 1,4,5-trisphosphate (Chuang, 1989). Though it has been shown that the peptide also activates phosphatidylinositol breakdown in rat uterine smooth muscle (Bousso-Mittler et al., 1989), we observed that ET-1 did not contract strips bathed in Ca:+-free solution. This result may indicate that, in the non-pregnant rat uterus, the intracellular Ca 2÷ pool available for mobilization by ET-1 was insufficient to evoke contraction per se. Nevertheless, such an action may have been responsible for the greater magnitude of contractions induced by ET-1 but not by Bay K 8644 in preparations challenged with the peptide in the absence of extracellular Ca 2÷ and subsequently exposed to the cation, as compared to those obtained with ET-1 added to matched strips bathed in normal De Jalon solution. Alternatively, this later observation could either reflect sensitization of the contractile machinery to Ca 2+ via protein kinase C activation, a sequence suggested to occur in aortic smooth muscle stimulated with ET-1 (Sugiura et al., 1989), or mean that Ca 2+ down-regulates ET receptors allosterically. However, Gu et al. (1989a) have shown that Ca 2÷ enhances rather than decreases the binding of ET-1 to rat cardiac membranes. Further studies are obviously needed to clarify this issue. Another interesting finding of the present study was that ET-1, given at a concentration insufficient to cause contraction per se, potentiated the contractile effects of PGE2~ and 5-HT in strips from non-pregnant rats and of All in late pregnant preparations. The mechanism(s) underlying this synergistic action of ET-1 remains unresolved, but could involve facilitation of Ca z+ channel opening a n d / o r of other intracellular transducing events. However, the potentiation of PGFz,-induced contractions by ET-1 could be particularly important for two reasons: (1) in humans, the large amounts of this eicosanoid generated by decaying endometrial cells during menses have been implicated in the etiology of dysmenorrhoea (Chan, 1983); and (2) ET-1, which is produced by endothelial cells stimulated with thrombin or other vasoconstrictors (Yanagisawa et al., 1988), triggers eicosanoid formation in several tissues (De Nucci et al., 1988; Rae et al., 1989; Payne and Whittle, 1988), induces hyperalgesia in rats and humans, pain in mice and synergizes with prostaglandin E 2 to cause long-lasting incapacitation when injected intra-articularly into
knee joints of dogs (Ferreira et al., 1989). Thus, ETs may have a physiological modulatory function in the regulation of uterine contractility and in the development of dysmenorrhoea. Furthermore, the occurrence of functional ET receptors in uterine smooth muscle and of specific high-affinity ET-1 binding sites in placental membranes (Fischli et al., 1989), as well as the demonstration that human umbilical vein endothelial cells can synthesize ET-1 (Fischli et al., 1989) and that placenta (Onda et al., 1990) can synthesize ET-3 may suggest a role for the ETs as triggers a n d / o r mediators of labour. It remains to be shown, however, whether uterine myometrium can also produce ETs. The current results extend the findings of Kosuka et al. (1989) and others which were published during the course of this study (Borges et al., 1989; Bousso-Mittler et al., 1989; Eglen et al., 1989) by demonstrating for the first time that ET-2, in addition to ET-1 and ET-3, contracts the uterus of the non-pregnant and late pregnant rat. The oxytocic potency of ET-1 is comparable to that of All and greater than that of ET-2 and ET-3 and of several important endogenous oxytocic agents such as OT, PGF2,, 5-HT and BK. The contractile activity of ET-1 is susceptible to modulation by sex hormones and is mediated by a mechanism(s) different from that of Bay K 8644. The potent direct contractile effect of ET-1 on myometrial tissue, allied to its ability to enhance contractions induced by other oxytocic agents suggest that this peptide may exert an important role in controlling the activity of non-pregnant and pregnant rat myometrium.
Acknowledgments We would like to express our gratitude to Miss Rosana Ostroski and Miss Adenir Pereira for their expert technical assistance and to Mrs Elizabet Ramos Ganzer for secretarial help. BAY K 8644 was a generous gift from Bayer A.G. (F.R.G.). This study receivedsupport from CNPq and FINEP (Brazil).
References Auguett, M., S. Delaflotte, P.E. Chabrier, E. Pirotzky, F. Clostre and P. Braquet, 1989, Endothelin and Ca+ + agonist Bay K 8644: different vasoconstrictive properties, Biochem. Biophys. Res. Conmaun. 156, 186. Borges, R., V. Von Grafenstein and D.E. Knight, 1989, Tissue selectivity of endothelin, European J. Pharmacol. 165, 223. Bousso-Mittler, D., Y. Kloog, Z. Wollberg,A. Bdolah, E. Kochva and M. Sokolovsky,1989, Functional endothelin/sarafotoxin receptors in the rat uterus, Biochem. Biophys. Res Commun. 162, 952. Chan, W.Y., 1983, Prostaglandins and nonsteroidal antiinflammatory drugs in dysmenorrhea, Ann. Rev. Pharmacol. Toxicol.23, 131. Chuang, D.-M., 1989, Neurotransmitter receptors and phosphoinsitide turnover, Ann. Rev. Pharmacol. Toxicol. 29, 71. De Nucci, G., R. Thomas, P. D'Orleans-Juste, E. Antunes, C. Walder, T.D. Warner and J.R. Vane, 1988, Pressor effects of circulating
116 endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor, Proc. Natl. Acad. Sci. U.S.A. 85, 9797. D'Orl6ans-Juste, P., G. De Nucci and J.R. Vane, 1989, Endothelin-1 contracts isolated vessels independently of dihydropyridine-sensitive Ca channel activation, European J. Pharmacol. 165, 289. Eglen, R.M., A.D. Michel, N.A. Sharif, S.R. Swank and R.L. Whiting, 1989, The pharmacological properties of the peptide endothelin, Br. J. Pharmacol. 97, 1297. Ferreira, S.H., M. Romitelli and G. De Nucci, 1989, Endothelin-1 participation in overt inflammatory pain, J. Cardiovasc. Pharmacol. 13 (Suppl. 5), $220. Fischli, W,, M. Clozel and C. Guilly, 1989, Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay, Life Sci. 44, 1429. Fuchs, A.R., 1978, Hormonal control of myometrial function during pregnancy and parturition, Acta Endocrinol. (Suppl. 221), 5. Fukuda, Y., Y. Hirata, H. Yoshimi, T. Kojima, Y. Kobayashi, M. Yanagisawa and T. Masaki, 1988, Endothelin is a potent secretagogne for atrial natriuretic peptide in cultured rat atrial myocytes, Biochem. Biophys. Res. Commun. 155, 167. Goto, K., Y. Kasuya, N. Matsuki, Y. Takuwa, H. Kurihara, T. Ishikawa, S. Kimura, M. Yanagisawa and T. Masaki, 1989, Endothelin activates the dihydropyridine-sensitive, voltage-dependent Ca 2+ channel in vascular smooth muscle, Proc. Natl. Acad. Sci. US.A. 86, 3915. Gu, X.H., D. Casley and W. Nayler, 1989a, Specific high-affinity binding sites for 125I-labelled porcine endothelin in rat cardiac membranes, European J. Pharmacol. 167, 281. Gu, X.H., J.J. Liu, J.J. Dilon and W.G. Nayler, 1989b, The failure of endothelin to displace bound, radioactively-labelled, calcium antagonists (PN 200/110, D888 and diltiazem), Br. J. Pharmacol. 96, 262. Hu, J.R., R. Von Harsdorf and R.E. Lang, 1989, Endothehn has potent inotropic effects in rat atria, European J. Pharmacol. 158, 275. Inoue, A., M. Yanagisawa, S. Kimura, Y. Kasuya, T. Miyauchi, K. Goto and T. Masaki, 1989, The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes, Proc. Natl. Acad. Sci. U.S.A. 86, 2863. Ishikawa, T., M. Yanagisawa, M. Kimura, K. Goto and T. Masaki, 1988, Positive chronotropic effects of endothelin, a novel endothelium-derived vasoconstrictor peptide, Pfliigers Arch. 413, 108. Kloog, Y. and M. Sokolovsky, 1989, Similarities in mode and sites of action of sarafotoxins and endothelins, Trends Pharmacol. Sci. 10, 212. Kosuka, M., T. Ito, S. Hirose, K. Takahashi and H. Hagiwara, 1989, 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 channels, Biochem. Biophys. Res. Commun. 159, 317. Komuro, Y., H. Kurihara, T. Sugiyama, F. Takaku and Y. Yazaki, 1988, Endothelins stimulate c-fos and c-myc expression and proliferation of vascular smooth muscle cells, FEBS Lett. 238, 249. Maggi, C.A., S. Giuliani, R. Patacchini, P. Santicioli, D. Turini, G. Barbanti and A. Meli, 1989b, Potent contractile activity of endothelin on the human isolated urinary bladder, Br. J. Pharmacol. 96, 755.
Maggi, C.A., R. Patacchini, S. Giuliani and S. Meli, 1989a, Potent contractile effect of endothelin in isolated gninea-pig airways, European J. Pharmacol. 160, 179. Moravec, C.S., E.E. Reynolds, R.W. Stewart and M. Bond, 1989, Endothehn is a positive inotropic agent in human and rat heart in vitro, Biochem. Biophys. Res. Commun. 159, 14. Onda, H., S. Okubo, K. Ogi, T. Kosawa, C. Kimura, H. Matsumoto, N. Suzuki and M. Fujino, 1990, One of the endothelin gene family, endothelin-3 gene, is expressed in the placenta, FEBS Lett. 261, 327. Payne, A.N. and B.J.R. Whittle, 1988, Potent cyclo-oxygenase-mediated bronchoconstrictor effects of endothelin in the guinea-pig in vivo, European J. Pharmacol. 158, 303. Rae, G.A., M. Tribulec, G. De Nucci and J.R. Vane, 1989, Endothelin-1 releases eicosanoids from rabbit isolated perfused kidney and spleen, J. Cardiovasc. Pharmacol. 13 (Suppl. 5), $89. Rakugi, H., M. Nakamaru, H. Saito, J. Higaki and T. Ogihara, 1988, Endothelin inhibits renin release from isolated rat glomeruli, Biochem. Biophys. Res. Commun. 155, 1244. Riemer, R.K. and J.M. Roberts, 1986, Endocrine modulation of myometrium response, in: The Physiology and Biochemistry of the Uterus in Pregnancy and Labor, ed. Gabor Huszar (CRC Press, Boca Raton) Ch. 5, p. 35. Silberberg, S.D., T.C. Poder and A.E. Lacerda, 1989, Endothelin increases single-channel currents in coronary arterial smooth muscle cells, FEBS Lett. 247, 68. Simonson, M.S., S. Wann, P. Mene, G.R. Dubyak, M. Kester, Y. Nakazato, J.R.A. Sedor and M.J. Dunn, 1989, Endothelin stimulates phospholipase C, N a + / H ÷ exchange, c-los expression, and mitogenesis in rat mesangial cells, J. Chn. Invest. 83, 708. Sugiura, M., T. Inagami, G.M.T. Hare and J.A. Johns, 1989, Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols, Biochem. Biophys. Res. Commun. 158, 170. Tabuchi, Y., M. Nakamaru, H. Rakugi, M. Nagano, H. Mikami and T. Ogihara, 1989, Endothelin inhibits presynaptic adrenergic neurotransmission in rat mesenteric artery, Biochem. Biophys. Res. Commun. 161, 803. Triggle, D.J. and D. Rampe, 1989, 1-4-Dihydropyridine activators and antagonists: structural and functional distinctions, Trends Pharmacol. Sci. 10, 507. Uchida, Y., H. Ninomiya, H. Ninotome, A. Nomura, M. Ohtsuka, M. Yanagisawa, K. Goto, T. Masaki and S. Hasegawa, 1988, Endothelin, a novel vasoconstrictor peptide, as potent bronchoconstrictor, European J. Pharmacol. 154, 227. Van Renterghem, C., P. Vigne, J. Barhanin, A. Schmidt-Alliana, C. Frelin and M. Ladzunski, 1988, Molecular mechanism of action of the vasoconstrictor peptide endothelin, Biochem. Biophys. Res. Commun. 157, 977. Watson, S. and A. Abbott, 1990, Receptor nomenclature supplement, Trends Pharmacol. Sci. 11 (Suppl.), 29. Wiklund, N.P., A. Ohlen and B. Cederqvist, 1988, Inhibition of adrenergic neuroeffector transmission by endothelin in the gnineapig femoral artery, Acta Physiol. Scand. 134, 311. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yasaki, K. Goto and T. Masaki, 1988, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332, 411.
109
EJP 51623
Effects of endothelins, Bay K 8644 and other oxytocics in non-pregnant and late pregnant rat isolated uterus Jo~o B. Calixto a n d Giles A. R a e Department of Pharmacology (CCB), Universidade Federal de Santa Catarina, 88049 Florian@olis, SC, Brazil Received 11 July 1990, accepted 25 September 1990
Endothelins 1, 2 and 3 (ET-1, ET-2 and ET-3; 1-30 nM) caused long-lasting concentration-dependent tonic contractions of uterine strips from non-pregnant rats. The potency of ET-1 (ECs0 7 nM) was similar to that of angiotensin II (AII) and greater than that of ET-2 or ET-3 (ECs0s >i 10 nM), bradykinin, Bay K 8644, oxytocin (OT), 5-hydroxytryptamine, prostaglandin F2, (PGF2~) or acetylcholine. Strips from 21-day pregnant rats were 2- to 3-fold more sensitive to ET-1, AII, OT and PGF2~ and 200-fold more sensitive to Bay K 8644 than non-pregnant preparations. The development of tonic responses to ET-1 (30 nM) and of phasic-rhythmic ones to Bay K 8644 (300 nM) was fully prevented in strips from non-pregnant rats bathed in Ca2+-free medium, but stepwise reintroduction of Ca :+ (0.03-3 mM) to the solution allowed the manifestation of contractions in response to both agonists. Responses to ET-1 required less Ca 2÷ than those to Bay K 8644. Strips challenged with ET-1 while in Ca2+-free medium developed greater contractions upon reintroduction of Ca 2+ than preparations stimulated with the peptide in normal medium. The reverse occurred with Bay K 8644-induced contractions. Nicardipine (10 nM) abolished the responses of strips from non-pregnant rats to Bay K 8644 (300 nM), but only attenuated ET-l-induced (30 nM) contraction. A subthreshold concentration of ET-1 (0.3 nM) augmented the responses to PGF2~ or 5-HT and to AII in preparations taken from non-pregnant and pregnant rats. Thus, ET-1 is one of the most powerful known constrictors of the rat isolated uterus. Its action is enhanced during late pregnancy and is mediated by a mechanism distinct from that of Bay K 8644. It is suggested that ETs may play significant roles in the control of uterine contractility. Endothelins; BAY k 8644; Oxytocic agents; Uterus (rat); Nicardipine; Pregnancy
1. Introduction
Recently, Yanagisawa et al. (1988) demonstrated that cultured porcine aortic endothelial cells release endothelin (ET) a 21-residue peptide of unusual structure with potent and long-lasting vasoconstrictor and pressor activities in vitro and in vivo, respectively. On a molar basis, the constrictor potency of ET on coronary artery rings isolated from pigs was found to be 100-fold greater than that of the potent vasoconstrictor, angiotensin II. It has since become evident (Inoue et al., 1989) that the human, rat and porcine genomes each contain three distinct isogenes encoding different ET-like peptides: ET-1 (previously named porcine ET), ET-2 and ET-3 (previously called rat ET). Also, these peptides show striking structural and pharmacological resemblance with the sarafotoxins present in the Atractaspis engaddensis snake venom (Kloog and Sokolovsky, 1989).
Correspondence to: J.B. Calixto, Department of Pharmacology, CCB, Universidade Federal de Santa Catarina, Ferreira Lima 26, 88015 Florian6polis, SC, Brasil.
Besides its potent vasoconstrictor effects (Yanagisawa et al., 1988; De Nucci et al., 1988), ET-1 causes positive inotropic and chronotropic effects on the heart (Hu et al., 1989; Ishikawa et al., 1988; Moravec et al., 1989), stimulates cell proliferation (Komuro et al., 1988; Simonson et al., 1989), triggers the release of eicosanoids (Rae et al., 1989) and atrial natriuretic peptide (Fukuda et al., 1988), inhibits renin release (Rakugi et al., 1988) and affects sympathetic neurotransmission (Wiklund et al., 1988; Tabuchi et al., 1989). Also, in vitro, ET-1 contracts non-vascular smooth muscle such as that of the rat stomach (De Nucci et al., 1988), guinea-pig trachea (Maggi et al., 1989a) human urinary bladder (Maggi et al., 1989b) and bronchi (Uchida et al., 1988). A recent study has also shown the marked oxytocic activity of ET-1 on rat uterine strips (Kosuka et al., 1989). Since the contractility of uterine smooth muscle is highly susceptible to modulation by sex hormones (Fuchs, 1978; Riemer and Roberts, 1986), the present study compared the effects of ET-1, ET-2 and ET-3 on the uterus isolated from non-pregnant and late pregnant rats with the effects of several known oxytocic agents. Also, because it was proposed that ET-1 is a putative
110 endogenous agonist of L-type voltage-dependent (dihydropyridine-sensitive) Ca 2+ channels (Yanagisawa et al., 1988) and was shown to strongly activate such channels (Goto et al., 1989; Silberberg et al., 1989), we compared its profile of activity with that of the dihydropyridine agonist, Bay K 8644. Finally, we assessed the influence of ET-1 on contractions induced by other oxytocic agents.
2. Materials and methods
2.1. Animals Adult female Wistar rats (180-250 g) kept in a room with controlled temperature (22 + 2°C) and illumination (12 h light and 12 h darkness) were used. Nonpregnant virgin animals were pretreated with oestradiol benzoate (0.5 mg/kg s.c.) 24 h before the experiments. Pregnant animals were obtained by pairing groups of 10 virgin female rats with three males overnight only. The rats that became pregnant were separated and killed 21 days later.
2.2. Experimental procedures The animals were killed by a blow on the head and their uteri were removed and carefully dissected free from adhering tissue. Pregnant uteri were cut transversally at points between foetus implants and the foetuses and placentas were gently removed. Uterine strips of 15 mm in length were suspended in 5-ml jacketed organ baths at 30 °C bubbled with air, containing physiological De Jalon solution of the following composition (mM): NaC1 154; KC1 5.6; CaCI 2 0.3; MgC12 1.4; NaHCO 3 1.7; and glucose 5.5. Isotonic contractions were measured under a resting load of 1 g using a light lever (six-fold amplification) writing on a kymograph. The tissues were equilibrated for at least 30-45 min before drug additions, during which time the bath solution was renewed every 15 min. After this period the preparations were first exposed to KC1 (80 mM)-rich solution (by equimolar replacement of 80 mM of NaC1 by KC1 in the medium). After washout, replacement with normal solution and complete relaxation (20-30 min), concentration-response curves were obtained to one of several agonists. Endothelins (ET-1, ET-2 and ET-3), Bay K 8644, bradykinin (BK), acetylcholine (ACh) and oxytocin (OT) were added to the bath in stepwise cumulative concentrations. Curves to 5-hydroxytryptamine (5-HT), angiotensin II (AII) and prostaglandin F2~ (PGF2~) were obtained by adding single increasing concentrations of the agonist to the bath for 30-60 s at 10-15 min intervals. The potencies of the various agonists were evaluated at the ECs0 level, i.e. at concentrations producing 50% of the maximal response.
Another set of experiments served to determine the influence of external Ca 2÷ on contractile responses of strips from non-pregnant rats to ET-1 (30 nM) and Bay K 8644 (300 nM). Following equilibration, the preparations were contracted with KC1 (80 mM) as before. The preparations were then bathed in Ca2+-free De Jalon solution containing 0.2 mM EGTA for 20 min, during which the solution was renewed every 5 min. This was followed by another 10 min in Ca2÷-free solution (without EGTA) before the addition of ET-1 (30 nM) or Bay K 8644 (300 nM) to the bath. After confirming the absence of a contractile response in Ca2÷-free solution and still in presence of the agonist, Ca 2÷ (0.03-3 mM) was added cumulatively to the bath. The contractions obtained in this way, i.e. by adding the agonist in Ca2+-free solution then reintroducing Ca 2+ to the bathing medium, were compared to those elicited by the agonist in time-matched strips taken from the same animals and bathed in normal solution (Ca 2÷ 0.3 mM) throughout the experiment. To analyse the possible participation of dihydropyridine-sensitive Ca 2÷ channels in the start of contractions induced by ET-1, uterine strips from non-pregnant rats were exposed to a single concentration of nicardipine (10 or 100 nM) and challenged 20 min later with ET-1 (30 nM) or Bay K 8644 (300 nM). The responses to either agonist obtained in the presence of the dihydropyridine antagonist were compared to those caused by ET-1 or Bay K 8644 alone in different, time-matched, strips from the same animals. We also evaluated the influence of nicardipine on established contractions induced by ET-1 (30 nM) or Bay K 8644 (300 nM). In these experiments cumulative concentrations of the Ca2÷-channel antagonist (10-100 nM) were added to the medium once responses to the agonists had stabilized and the degree of inhibition was measured 20 min after each addition. The influence of ET-1 on the contractile responses of strips from non-pregnant and late pregnant rats to other agonists was assessed in a separate sequence of experiments. After a response to KC1 (80 nM) had been obtained, the preparations were stimulated repeatedly at 10- to 15-min intervals with a submaximally effective concentration (EC40 to EC70) of a given agonist. Once the contractions became reproducible, ET-1 (0.3 mM) was added to the bath for 10 rain and a new contraction in response to the agonist was obtained in its presence. In all experiments the contraction induced by KC1 (80 mM) was taken as the 100% response and the effects of the various agonists are presented as functions of this value.
2.3. Statistics The ECs0s are presented as geometric means accompanied by their 95% confidence limits. All other values
111
are presented as means + S.E.M. Statistical analyses were performed by means of two-tailed paired and unpaired Student's t-tests where appropriate, and P < 0.05 was considered significant.
3. Results
3.1. Effects of endothelins and Bay K 8644 on uterine strips from non-pregnant and pregnant rats
2.4. Drugs The drugs used were: ET-1 (porcine), ET-2 (human) and ET-3 (rat; all from the Peptide Institute Inc., Japan), Bay K 8644 (methyl-l,4-dihydro-2,6-dimethyl-3-nitro-4(2-tri-fluoromethylphenyl)-pyridine-5-carboxylate from Bayer A.G., FRG), bradykinin, acetylcholine iodide, 5-hydroxytryptamine creatinine sulphate, nicardipine, oestradiol benzoate (all from Sigma Chemical Company, USA), prostaglandin F2~ (Chinoin, Hungary), oxytocin (Syntocinon~, Sandoz, Brazil) and angiotensin II (synthesized by the Department of Biophysics, Escola Paulista de Medicina, $5o Paulo, Brazil). Most drugs were stored as 1-100 mM stock solutions at - 2 0 ° C and diluted to the desired concentration in PBS solution just before use. Endothelins were diluted in PBS solution from 10/~M stock solutions stored at - 2 0 ° C. The stock solutions of Bay K 8644 and nicardipine (10 raM) were made up in absolute ethanol and all others were prepared in PBS solution. Oestradiol was dissolved in peanut oil to 1 mg/ml. Solutions containing Bay K 8644 or nicardipine were protected from light and the experiments were carried out in the dark with these compounds to avoid photodegradation.
Cumulative additions of ET-1, ET-2 and ET-3 (0.1-30 nM) caused concentration-dependent tonic contractions of both non-pregnant and pregnant rat uterine strips, which were only slowly reversible following washout. Contractions induced by the highest concentration of ET-1 or ET-2 (30 nM) failed to subside completely within 60 min of washout, despite solution renewal every 10-15 min. The mean results depicted in fig. 1A-C and summarized in table 1 show that ET-1 was more potent than ET-2 and ET-3 to cause contractions of non-pregnant uterine strips. Although complete concentration-response curves for the ETs were not obtained due to limited availability of the peptides, contractions caused by the highest ET-1 concentration tested (30 nM) were comparable to those elicited by KC1 (80 mM) and significantly greater than responses to ET-2 or ET-3 (30 nM). The contractile concentration-response curve to ET-1 with 21-day pregnant rat uterine strips was shifted to the left about 2-fold relative to that obtained in preparations from non-pregnant animals (P < 0.05), as evaluated at the apparent ECs0 level (fig. 1A and table 1). ET-2 and ET-3 also seemed to be more potent in
TABLE 1 Responsiveness of non-pregnant and late pregnant (21 days) rat isolated uterus to endothelins 1, 2 and 3, Bay K 8644 and other oxytocic agents. Agonist
Endothelin-I Endothelin-2 Endothelin-3 Bay K 8644 Angiotensin II Bradykinin Serotonin Prostaglandin F2,~ Acetylcholine Oxytocin
Non-pregnant
Late pregnant
ECs0"
Maximal b response
ECs0
6.9 nM (5.3-8.8) >~10 nM >~10 nM 21.2 nM (14.5-39.1) 8.8 nM (6.7-11.6) 16.5 nM (10.4-26.2) 0.3/zM (0.1-0.5) 1.0/~M (0.7-1.5) 1.0 #M (0.7-1.4) 50.0 nM (37.1-62.5)
102.5_+ 7.5 c
3.8 nM d (2.7-5.2) >i 10 nM t> 10 nM 0.1 nM d (0.04-0.3) 2.6 nM a (1.8-3.8) 6.5 nM d (1.9-21.7) 0.4 ~M (0.2-0.6) 0.3/.tM d (0.2-0.7) 1.0 ~M (0.7-1.5) 16.7 nM d (12.5-25.0)
70.3-+ 5.4 c 21.9 + 12.8 ¢ 119.4+ 5.8 99.7+ 2.0 122.1-+ 5.5 116.7_+ 6.6 116.1 + 5.3 118.5 + 4.5 110.1 + 7.2
Maximal response 86.4-1- 5.9 c 62.45- 5.8 c 35.4 + 9.2 c 106.2+ 3.4 d 199.2+ 9.5 66.2+ 8.3 d 90.9+ 9.2 d 100.0+ 11.1 141.9 + 11.0 d 118.0+ 7.5
" ECs0s presented as the geometric means accompanied by 95% confidence limits, b Calculated as percentages of the response to KCI 80 mM (mean-+ S.E.M. of five to eight observations), c Response to highest concentration tested (30 nM). d p < 0.05 when compared to non-pregnant group.
112 i20'
Bay K 8644 (1-1000 nM) caused concentration-dependent phasic contractions of the non-pregnant rat uterus which were relatively resistant to washout. The pattern of contractions induced by Bay K 8644 was markedly different from that obtained with ET-1 (compare figs. 4A and C). Also in contrast to endothelin-induced contractions, the responses to Bay K 8644 were markedly influenced by pregnancy, as evidenced by an about 200-fold shift of the concentration-response curve at the ECs0 level and a slight but significant reduction of the maximal response (fig. 1D and table 1). Thus, Bay K 8644 was about 3-fold less potent than ET-1 in preparations from non-pregnant rats, but nearly 40-fold more potent than the peptide in strips from late pregnant animals.
B
A
tOO" O NON-PREGNANT /.~
£z
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80-
Q PREGNANT-
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2t
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o ~
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3.2. Contractions of the non-pregnant and late pregnant rat uterus induced by other oxytocics
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0t O.i i 10 tO0 1000 ENDOTHELIN-5 (nM) BAY K 8 6 4 4 (n M} Fig. 1. Mean contractile concentration-response curves for endothelin1 (A), endothelin-2 (B), endothelin-3 (C) and for Bay K 8644 (D) in the isolated uterus from 21-day pregnant (e) and non-pregnant (o) rats. Each point represents the mean of five to eight experiments from at least two different animals and the vertical bars indicate the S.E.M.
The mean contractile concentration-response curves for AII, BK, 5-HT, PGF2~, ACh and OT are illustrated in fig. 2. All the agonists caused concentration-dependent contractions of the non-pregnant uterus with the following rank order of potency: A I I > BK > OT > 5HT > PGF2~ = ACh (table 1). In preparations isolated from late pregnant rats, the contractile concentrationresponse curves for AII, PGF2~ and OT were significantly shifted to the left about 3-fold (P < 0.05), while the sensitivities to BK, ACh and 5-HT did not differ
pregnant strips, but an accurate estimation of this phenomenon was again hampered by the impossibility of making complete curves (fig. 1B, C and table 1). t40"
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Fig. 2. Mean contractile concentration-response curves for angiotensin II (A), bradykinin (B), serotonin (C), prostaglandin F2~ (D), acetylcholine (E) and oxytocin (F) in the uterus isolated from 21-day pregnant (e) and non-pregnant (0) rats. Each point represents the mean of 6 to 10 experiments with preparations from at least two different animals and the vertical bars indicate the S.E.M.
113 f r o m those of strips from n o n - p r e g n a n t rats (table 1), yielding the rank order of potency: A I I > B K > O T > 5 - H T = PGF2~ > ACh. Maximal contractile responses to the various agonists were also differentially affected by pregnancy (table 1). Whereas the maximal contractions induced by A I I and A C h were enhanced significantly, those induced b y BK and 5 - H T were reduced (P < 0.05). The maximal responses to PGFz~ and O T were similar in tissues from pregnant and n o n - p r e g n a n t rats.
ENDOTHELIN- t (,30 nM ) •
BAY K 8 6 4 4 (300 nM ) li,
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3.3. Influence of extracellular calcium content and nicardipine on effects of endothelin-1 and Bay K 8644 C"
Uterine preparations from non-pregnant rats, after equilibration for 30 min in Ca2+-free m e d i u m that contained 0.2 m M E G T A for the first 20 min, were completely refractory to the contractile effects of ET-1 (30 nM) and Bay K 8644 (300 nM). However, subsequent stepwise reintroduction of Ca 2+ (0.01 to 3 m M ) to the bathing solution, in the presence of either substance, p r o m p t l y restored their contractile effects in a concentration-related m a n n e r (fig. 3). In such experiments, the tonic response to ET-1 (fig. 3B) was about 3-fold more sensitive to Ca z+ reintroduction, with ECs0 0.20 m M (0.12-0.30, n = 5), than the phasic-rhythmic response to Bay K 8644, ECs0 0.63 m M (0.47-8.5, n = 7) (fig. 3D). Also, the strips challenged with ET-1 in CaZ+-free m e d i u m then exposed to increasing extracellular Ca 2+ concentrations (0.3-3 raM) showed significantly greater responses than time-matched preparations stimulated with the peptide while in normal D e Jalon solution (0.3 m M Ca 2+) (compare figs. 3A and B). In contrast, preparations challenged with Bay K 8644 in Ca2+-free solution exhibited contractions u p o n Ca 2+ reintroduction (fig. 3D) which were significantly
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CoCI2 CONCENTRATION (raM) Fig. 4. Influence of extracellular calcium concentration on the magnitude of contractions induced by endothelin-1 (30 nM) and Bay K 8644 (300 nM) in the non-pregnant rat isolated uterus. The preparations were exposed to the agonists either in Ca2+-free medium (open columns) or in normal De Jalon solution containing 0.3 mM Ca 2÷ (hatched columns). Responses were recorded in presence of the extracellular Ca 2+ concentrations indicated in the figure. Each column represents the mean of five to eight experiments and the vertical bars indicate the S.E.M. The responses to either agonist, added in the presence or absence of extracellular Ca 2÷, were significantly different at any given concentration of Ca 2+ (P < 0.05).
smaller than those obtained in strips bathed in normal De Jalon solution at the time of agonist challenge (fig. 3C). The means for the results of these experiments are presented in fig. 4. It is also noteworthy that, in the records illustrated in figs. 3C, D, with concentrations of Ca 2+ in excess of 0.3 mM, the responses to Bay K 8644 exhibited an additional tonic component. Preincubation of strips f r o m n o n - p r e g n a n t rats with nicardipine (10-
20 rain
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Fig. 3. Representative isotonic tracings for the influence of extracellular calcium concentration on contractions induced by endothelin-1 (ET-1, 30 nM) or Bay K 8644 (300 nM) in non-pregnant rat isolated uterus. Note that in the experiments of panels A and C the agonists were added to strips bathed in normal De Jalon solution containing 0.3 mM Ca 2÷. In the experiments of panels B and C the preparations were equilibrated for 30 min with Ca2+-free solution (plus EGTA 0.2 mM for the first 20 min) and challenged with the agonist in CaZ+-free medium. Cumulatively increased concentrations of Ca 2+ were added to the bath as indicated in the figure. Similar results were obtained in another four experiments.
114 NON-PREGNANT
iO0" Z
pregnant rats to BK interfered with the analysis of the influence of ET-1 on BK-induced responses. Also, we were unable to analyse possible interactions between ET-1 and Bay K 8644 in strips from either non-pregnant or pregnant rats, because of the very slow reversibility of contractions caused by the dihydropyridine.
[ ] CONTROL
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Fig. 5. Influence of prcincubation with endothe]Jn-] (0.3 n]Vl) on the
contractile response induced by angiotensin II (AII), serotonin (5-HT), prostaglandin F2~ (PGF2~), acetylcholine (ACh), oxytocin (OT) and bradykinin (BK) in the non-pregnant (A) and 21-day pregnant (B) rat isolated uterus. Open columns represent control responses and hatched columns indicate the responses obtained in the presence of endothelin-1. Each column represents the mean of five to eight experiments and the vertical lines indicate the S.E.M. Endothelin-1 significantly enhanced the responses of strips from non-pregnant rats to 5-HT and PGF2~ and of 21-day pregnant strips to All (P < 0.05).
100 nM) for 20 rain caused partial graded inhibition of the development of responses to ET-1 (30 nM). At 10 and 100 nM, nicardipine reduced the height of contractions caused by ET-1 from 126.8 + 5.6% of the response to KC1 80 mM to 51.7 + 7.4 and 39.2 + 10.4%, respectively (n = 5). Established contractions induced by ET-1 were somewhat less susceptible to inhibition, so that about half of the original control response persisted in the presence of 100 nM of the dihydropyridine antagonist (59.8 + 7.5% of the response to KC1 80 mM, n = 5). In contrast, nicardipine at 10 nM fully abolished both the start and maintenance of Bay K 8644-induced responses (300 nM, n = 6). 3.4. Influence of endothelin-1 on contractions induced by
other oxytocics Figure 5A shows that incubation of non-pregnant rat uterine strips for 10 rain with a low concentration of ET-1 (0.3 nM), which alone did not cause contraction, significantly potentiated the responses to submaximally effective concentrations of 5-HT (10-100 nM) or PGF2~ (1/~M). However, the presence of ET-1 (0.3 nM) failed to alter contractions elicited by All (3 nM), BK (3 nM), ACh (1/xM) or OT (120 nM). In preparations from late pregnant rats, ET-1 potentiated only the responses to All (fig. 5B). The marked tachyphylaxis of strips from
4. Discussion
The results of the present study demonstrated that ET-1, ET-2 and ET-3 cause longqasting and concentration-dependent tonic contractions of uterus isolated from the non-pregnant rat. The rank order of potency was ET-1 > ET-2 >~ ET-3. On a molar basis, the potency of ET-1 was similar to that of All and greater than that of BK, Bay K 8644, OT, 5-HT, PGF2~ and ACh. Preparations isolated from rats on the 21st day of gestation were 2- to 3-fold more sensitive to ET-1, AII, OT and PGF2~ than strips from non-pregnant animals, whereas the sensitivity to Bay K 8644 was increased 200-fold. These results indicate that ET-1 is a remarkably potent oxytocic agent and that its action, like that of several (but not all) other agonists in this tissue, is susceptible to hormonal modulation. The markedly different patterns of contraction induced by ET-1 and Bay K 8644 in non-pregnant rat uterine strips further substantiate the view that ETs do not act as initially proposed (Yanagisawa et al., 1988) by direct activation of voltage-dependent L-type Ca 2+ channels. Bay K 8644, a dihydropyridine which activates such channels directly, caused phasic-rhythmic contractions, similar to those caused by OT. The rhythmicity of these responses may have been determined by intermittent or recurrent opening of high conductance C a 2+activated potassium channels (Watson and Abbott, 1990), leading to transient reductions of the ability of Bay K 8644 to activate L-type Ca 2+ channels (Triggle and Rampe, 1989). In contrast, ET-1 caused typically tonic contractions which, unlike those triggered by Bay K 8644, were only partially blocked by high concentrations of nicardipine. The finding that both ET-1 and Bay K 8644 failed to contract preparations bathed in CaE+-free solution indicates that responses to either agonist depended critically on the influx of Ca 2+ from the extracellular medium. Therefore, as previously demonstrated with vascular smooth muscle (Van Renterghem et al., 1988), it appears that uterine contractions induced by ET-1 involved primarily the influx of Ca 2+ through nicardipine-insensitive non-selective cation channels. The ensuing depolarization would then lead to activation of voltage-dependent L-type C a 2+ channels, accounting for the nicardipine-sensitive component of the ET-1 action. Taken together, the current data are consistent with reports showing that ET-1 fails to displace radio-labelled dihydropyridine antagonists
115 bound to smooth muscle membranes (Gu et al., 1989b), that it activates L-type Ca 2÷ channels indirectly (Silberberg et al., 1989) by opening non-selective cation channels (Van Renterghem et al., 1988) and causes contractions which are distinct from those induced by Bay K 8644 in isolated blood vessels (Auguet et al., 1988; D'Odrans-Juste et al., 1989). In vascular smooth muscle ET-1 stimulates phosphoinositide metabolism and mobilizes intracellular Ca 2÷ stores (Van Renterghem et al., 1988), possibly by raising the cytoplasmic levels of inositol 1,4,5-trisphosphate (Chuang, 1989). Though it has been shown that the peptide also activates phosphatidylinositol breakdown in rat uterine smooth muscle (Bousso-Mittler et al., 1989), we observed that ET-1 did not contract strips bathed in Ca:+-free solution. This result may indicate that, in the non-pregnant rat uterus, the intracellular Ca 2÷ pool available for mobilization by ET-1 was insufficient to evoke contraction per se. Nevertheless, such an action may have been responsible for the greater magnitude of contractions induced by ET-1 but not by Bay K 8644 in preparations challenged with the peptide in the absence of extracellular Ca 2÷ and subsequently exposed to the cation, as compared to those obtained with ET-1 added to matched strips bathed in normal De Jalon solution. Alternatively, this later observation could either reflect sensitization of the contractile machinery to Ca 2+ via protein kinase C activation, a sequence suggested to occur in aortic smooth muscle stimulated with ET-1 (Sugiura et al., 1989), or mean that Ca 2+ down-regulates ET receptors allosterically. However, Gu et al. (1989a) have shown that Ca 2÷ enhances rather than decreases the binding of ET-1 to rat cardiac membranes. Further studies are obviously needed to clarify this issue. Another interesting finding of the present study was that ET-1, given at a concentration insufficient to cause contraction per se, potentiated the contractile effects of PGE2~ and 5-HT in strips from non-pregnant rats and of All in late pregnant preparations. The mechanism(s) underlying this synergistic action of ET-1 remains unresolved, but could involve facilitation of Ca z+ channel opening a n d / o r of other intracellular transducing events. However, the potentiation of PGFz,-induced contractions by ET-1 could be particularly important for two reasons: (1) in humans, the large amounts of this eicosanoid generated by decaying endometrial cells during menses have been implicated in the etiology of dysmenorrhoea (Chan, 1983); and (2) ET-1, which is produced by endothelial cells stimulated with thrombin or other vasoconstrictors (Yanagisawa et al., 1988), triggers eicosanoid formation in several tissues (De Nucci et al., 1988; Rae et al., 1989; Payne and Whittle, 1988), induces hyperalgesia in rats and humans, pain in mice and synergizes with prostaglandin E 2 to cause long-lasting incapacitation when injected intra-articularly into
knee joints of dogs (Ferreira et al., 1989). Thus, ETs may have a physiological modulatory function in the regulation of uterine contractility and in the development of dysmenorrhoea. Furthermore, the occurrence of functional ET receptors in uterine smooth muscle and of specific high-affinity ET-1 binding sites in placental membranes (Fischli et al., 1989), as well as the demonstration that human umbilical vein endothelial cells can synthesize ET-1 (Fischli et al., 1989) and that placenta (Onda et al., 1990) can synthesize ET-3 may suggest a role for the ETs as triggers a n d / o r mediators of labour. It remains to be shown, however, whether uterine myometrium can also produce ETs. The current results extend the findings of Kosuka et al. (1989) and others which were published during the course of this study (Borges et al., 1989; Bousso-Mittler et al., 1989; Eglen et al., 1989) by demonstrating for the first time that ET-2, in addition to ET-1 and ET-3, contracts the uterus of the non-pregnant and late pregnant rat. The oxytocic potency of ET-1 is comparable to that of All and greater than that of ET-2 and ET-3 and of several important endogenous oxytocic agents such as OT, PGF2,, 5-HT and BK. The contractile activity of ET-1 is susceptible to modulation by sex hormones and is mediated by a mechanism(s) different from that of Bay K 8644. The potent direct contractile effect of ET-1 on myometrial tissue, allied to its ability to enhance contractions induced by other oxytocic agents suggest that this peptide may exert an important role in controlling the activity of non-pregnant and pregnant rat myometrium.
Acknowledgments We would like to express our gratitude to Miss Rosana Ostroski and Miss Adenir Pereira for their expert technical assistance and to Mrs Elizabet Ramos Ganzer for secretarial help. BAY K 8644 was a generous gift from Bayer A.G. (F.R.G.). This study receivedsupport from CNPq and FINEP (Brazil).
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