# In vivo pharmacological characterization of alpha adrenergic receptors in sheep myometrium and their physiological meaning

Pierre-Guy Marnet, Rafael Garcia Villar, Michel Laurentie, P.L. Toutain

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Pierre-Guy Marnet, Rafael Garcia Villar, Michel Laurentie, P.L. Toutain. In vivo pharmacological characterization of alpha adrenergic receptors in sheep myometrium and their physiological meaning. Biology of Reproduction, 1987, 37 (1), pp.241-248. 10.1095/biolreprod37.1.241. hal-02719421

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# In Vivo Pharmacological Characterization of Alpha Adrenergic Receptors in Sheep Myometrium and Their Physiological Meaning<fnref n="1" />

P. G. MARNET, R. GARCIA-VILLAR,
M. P. LAURENTIE, and P. L. TOUTAIN

Station de Pharmacologie-Toxicologie,
INRA
31300 Toulouse, France

## ABSTRACT

Utero-cervical responsiveness to alpha-adrenergic receptor stimulation was electromyographically evaluated in vivo in adult ovariectomized ewes. Spontaneous motility and drug-induced changes were quantified with a microcomputer.

Our results suggested that the two activity patterns previously described on the ovine genital tract could be respectively controlled by alpha-2 and alpha-1 adrenergic mechanisms. Alpha-2 receptors could be assigned to a chronotropic function by activating a hypothetical zone triggering the outbreak of discrete episodes of motility (regular activity); this function is apparent at the cervix even in the absence of estrogen priming. On the other hand, alpha-1 receptors could be assigned to an inotropic function, regulating mainly the magnitude of estrogen-dependent utero-cervical motility (irregular activity).

## INTRODUCTION

In several species, including sheep, the uterus contains both alpha ($\alpha$) and beta ($\beta$) adrenoreceptors mediating, respectively, excitatory and inhibitory responses to catecholamines (Alhquist, 1948; Marshall, 1970). Pharmacological properties and physiological significance of uterine beta receptors are well known due to extensive therapeutic use of $\beta$-2 agonists. In contrast, corresponding information concerning alpha receptors is rather scarce. By studying the displacement of ³H-dihydroergocriptine from rabbit uterine cell membrane by prazosin and yohimbine, it was clearly demonstrated that both subtypes of adrenoreceptors were present in the rabbit uterus (Hoffman et al., 1981). In some other species, presence of both $\alpha$-1 and $\alpha$-2 adrenoreceptors is probable. In the cyclic sow, it was shown that most of the uterine alpha receptors were of the $\alpha$-2 subtype (Rexroad and Guthrie, 1983). In sheep, uterine alpha receptors display a lower affinity for phenylephrine, a

potent $\alpha$-1 agonist, than for epinephrine and nor-epinephrine, which share both $\alpha$-1 and $\alpha$-2 potencies (Rexroad, 1981), suggesting that $\alpha$-2 is also the dominant subtype of sheep uterine alpha receptors.

Uterine alpha receptors are subject to regulation by ovarian steroids, but variations exist among species. In rabbit, estrogen treatment selectively increased $\alpha$-2 receptors (Hoffman et al., 1981). Conversely, progesterone treatment of ovariectomized ewes increased the concentration of alpha receptors (Rexroad, 1981), which were probably of the $\alpha$-2 subtype. Similarly, in the pig, $\alpha$-2 receptors were more numerous during the luteal phase (Rexroad and Guthrie, 1983).

As for the control of alpha receptor population by steroid hormones, the physiological meaning of the two subtypes of alpha receptors remains controversial. In the rabbit, it was shown that only $\alpha$-1 receptors were coupled to contractile responses, whereas $\alpha$-2 receptors were not directly implicated in uterine contractions (Hoffman et al., 1981); this explained why estrogen treatment did not change the sensitivity of the myometrium in this species to agonist-induced contractions that were mediated by $\alpha$-1 receptors. These findings contrasted with those reported in the same species by Roberts et al. (1981) who concluded that estrogen increased both populations of alpha receptors and responsiveness of the uterus.

<footnote>Accepted February 16, 1987.</footnote>

<footnote>Received July 28, 1986.</footnote>

<footnote n="1">¹This work was taken from a chapter of Dr. P. G. Marnet's Doctoral Thesis.
²Reprint requests: Dr. R. Garcia-Villar, Station de Pharmacologie-Toxicologie, I.N.R.A., 180 chemin de Tournefeuille, 31300 Toulouse, France.</footnote>
In intact cyclic or ovariectomized estradiol-treated ewes, phenylephrine or methoxamine, which display a selective affinity for α-1 receptors, increases uterine contractions (Hawk and Conley, 1985; Prud'homme, 1986); on the other hand, xylazine, an imidazoline derivative close to clonidine and which is predominantly an α-2 agonist, had demonstrated a clear-cut oxytocic effect in the late pregnant ewe (Janssen et al., 1984). Therefore, both types of alpha-receptor agonists lead to a stimulatory response of the uterus. Nevertheless, preliminary observations of our group have shown that uterine activations by either α-1 or α-2 agonists were not qualitatively equivalent, each type of agonist leading to the preferential occurrence of one of the two patterns of uterine activity that have been described previously (Garcia-Villar et al., 1982; Toutain et al., 1985), i.e. regular activity (α-2 agonists) and irregular activity (α-1 agonists). The objectives of the present work were to qualify the nature of the α-1 vs. α-2 adreno-receptor-induced uterine activations and their control by estrogens, and to suggest a hypothesis concerning their possible involvement in the regulation of physiological utero-cervical activity patterns.

## MATERIALS AND METHODS

The experiments were conducted during autumn and winter in 4 adult ewes of the Lacaune breed. Animals (58 ± 7 kg) were placed in individual cages and fed hay and oats ad libitum. Under aseptic surgical conditions and thiopentone anesthesia, animals were ovariectomized and fitted with intraparietal Ni-Cr wire electrodes, positioned on the ventral side of the cervix and on the middle of each uterine horn. Recordings started 2 days after surgery and the first experiment not before 3-4 wk after surgery, i.e. after completion of the typical pattern of activity described after ovariectomy (Garcia-Villar et al., 1982). Until the end of the experimental session (2-3 mo later), the electrodes were connected to the input pannel of a direct writing electromyographic amplifier (Reega Minihuit, Alvar, France). Direct records were taken randomly while, as a monitoring procedure, continuous (24 h/day) integration of the output signals was performed (Latour, 1973). Signals, displayed on a potentiometric recorder, were automatically quantified using an on-line microcomputer.

The first part of the study was designed to describe, qualitatively, the response pattern of the uterus and

cervix according to the activated alpha receptor. Adrenaline (which shares both α-1 and α-2 potencies) was infused after beta receptor blockade to test the overall response to alpha receptor simulation; α-1- and α-2-receptor-mediated responses were then demonstrated by the use of selective α-2 and α-1 antagonists, respectively. Adrenaline was infused for 20 min (1 µg/kg/min) in the jugular vein by means of a portable syringe driver (MS16, Graseby Medical, UK) through an indwelling catheter. All infusions started 10 min after a single i.v. injection of propranolol; the dosage of propranolol was increased during the assay from 0.05 to 0.1 mg/kg to overcome the development of a down-regulation at the beta receptor level. With the appropriate dosage regimen, propranolol blocked beta receptor response for 1 to 2 h. The α-1 mediated effect of adrenaline was evidenced after yohimbine (1 mg/kg) or tolazoline (1 mg/kg) administration. Under the same conditions, α-2-mediated effects of adrenaline were evidenced after administration of prazosin (1 mg/kg) or thymoxamine (1 mg/kg). Antagonists were administered by venipuncture in the opposite jugular vein 5 min before adrenaline infusion. In our experimental conditions, all of the antagonists used, including propranolol, were devoid of effect on utero-cervical motility. In addition to the adrenaline test, 20-min infusions of specific α-1 agonists, i.e., phenylephrine (5 µg/kg/min) or methoxamine (0.4 mg/kg/min), and α-2 agonists, i.e. clonidine (0.3 µg/kg/min) or xylazine (1 µg/kg/min), were used to assess both alpha effects. Commercial solutions were used directly; other drugs were dissolved in distilled water, except prazosin, which was dissolved in dimethylsulfoxide (DMSO; 5 ml in toto), and yohimbine, which was dissolved in DMSO and distilled water (50/50 to a final volume of 5 ml).

In the second part of the study, a cross-over-designed experiment was undertaken to quantify the development of alpha receptor responsiveness in the uterus, according to the time elapsed after a single i.m. administration of 17β-estradiol (2 µg/kg in ethanol). Adrenaline infusions were performed, as described above, before and regularly after estrogen priming. Thymoxamine and tolazoline, respectively, were selected as α-1 and α-2 antagonists and were administered as described above. The general schedule of this second assay is summarized in Table 1. Two series of infusions were performed at 3-wk intervals.

For this second assay, uterine myoelectrical
<table><caption>TABLE 1. General schedule of drug administration for the quantitative study.</caption><thead><tr><th colspan="3">Time according to estrogen (h)</th><th colspan="3">First experiment series</th><th colspan="3">Second experiment series</th></tr><tr><th>(1)*</th><th>(2)</th><th>(3)</th><th>(1)</th><th>(2)</th><th>(3)</th><th>(1)</th><th>(2)</th><th>(3)</th></tr></thead><tbody><tr><td>-24</td><td>-20</td><td>-16</td><td>A<sup>++</sup></td><td>A2</td><td>A1</td><td>A</td><td>A1</td><td>A2</td></tr><tr><td>2.5</td><td>3.5</td><td>-</td><td>A</td><td>A1</td><td>-</td><td>A</td><td>A2</td><td>-</td></tr><tr><td>5</td><td>6</td><td>-</td><td>A</td><td>A2</td><td>-</td><td>A</td><td>A1</td><td>-</td></tr><tr><td>7.5</td><td>8.5</td><td>-</td><td>A</td><td>A1</td><td>-</td><td>A</td><td>A2</td><td>-</td></tr><tr><td>10</td><td>11</td><td>-</td><td>A</td><td>A2</td><td>-</td><td>A</td><td>A1</td><td>-</td></tr><tr><td>24</td><td>28</td><td>32</td><td>A</td><td>A1</td><td>A2</td><td>A</td><td>A2</td><td>A1</td></tr><tr><td>48</td><td>52</td><td>56</td><td>A</td><td>A2</td><td>A1</td><td>A</td><td>A1</td><td>A2</td></tr><tr><td>72</td><td>76</td><td>80</td><td>A</td><td>A1</td><td>A2</td><td>A</td><td>A2</td><td>A1</td></tr><tr><td>96</td><td>100</td><td>104</td><td>A</td><td>A2</td><td>A1</td><td>A</td><td>A1</td><td>A2</td></tr><tr><td>120</td><td>124</td><td>128</td><td>A</td><td>A1</td><td>A2</td><td>A</td><td>A2</td><td>A1</td></tr><tr><td>168</td><td>172</td><td>176</td><td>A</td><td>A2</td><td>A1</td><td>A</td><td>A1</td><td>A2</td></tr><tr><td>216</td><td>220</td><td>224</td><td>A</td><td>A1</td><td>A2</td><td>A</td><td>A2</td><td>A1</td></tr></tbody></table>

activity was quantified every 20 sec by the on-line microcomputer. Mean values were automatically calculated for periods of 5 min and expressed in arbitrary computer units. Spontaneous activity (control) levels were measured during the hour that preceded each test infusion. Activity levels during infusions were considered only after the full response was reached, i.e. only for the last 15 min of infusion. For each animal and for each of the two periods, the highest observed response to the adrenaline test was recorded as 100% response. Other responses were rated as percentages of this maximal response (see Table 2). Kinetic analyses of data were done with a personal computer using a program for non-linear regression analysis (Koeppé and Hamann, 1980). Initial estimates were obtained using linear regression methods.

### Drugs

The drugs used were adrenaline (ADRENALINE, Aguettant, Lyon, France); propranolol (AVLO-CARDYL, ICI Pharma, Enghien, France); yohimbine hydrochloride (Sigma Chemical Co., St. Louis, MO); tolazoline hydrochloride (Sigma Chemical Co.); prazosin hydrochloride (Pfizer Inc. Orsay, France); thymoxamine (MOXISYLYTE, Dedieu, Bordeaux, France); phenylephrine (NEOSYNEPHRINE, Boehringer Ingelheim, Reims, France); methoxamine (VASO-XYL, Wellcome, London, England); clonidine (CATA-PRESSAN, Boehringer Ingelheim); xylazine (ROM-PUN, Bayer, Leverkusen, G.F.R); dimethylsulph-

oxyde (Farmitalia Carlo-Erba, Milan, Italy); and 17β-estradiol (Sigma Chemical Co.).

## RESULTS

### Qualitative Study

1.) Study before estradiol priming. Within 3 wk postsurgery and in the absence of estradiol priming, the uterine horns became quiescent, whereas the cervix continued to present regular episodes of electrical activity of 6 to 8-min duration at intervals of 1 to 2 h, which previously were termed regular spiking activity and shown to be related to episodes of muscular contraction (Garcia-Villar et al., 1982). Experiments were begun 1 wk after this stable pattern was established.

Under these conditions, when adrenaline was infused during a 20-min period between 2 episodes of regular activity and after the blockade of beta receptors by propranolol, a motor response restricted to the cervix was observed. It consisted of an episode of electrical activity lasting 12.91 ± 3.38 min (mean ± SD; n = 23), ending always before the 20-min infusion was completed. This cervical response was blocked by prior administration of either tolazoline or yohimbine, but not by thymoxamine or prazosin.

Similarly, a 20-min infusion of clonidine or xylazine elicited a discrete response on the cervix, in the form of a succession of episodes of regular spiking activity occurring at a frequency higher than in the controls during 1 to 4 hours (Fig. 1). Such responses were

<footnote>+Three adrenaline infusions (noted 1, 2, 3) were performed 4 h apart, each day, except within the day of estradiol injection: 4 pairs of infusions.</footnote>

<footnote>++A: Adrenaline alone (overall α effect); A1: adrenaline after tolazoline (α-1 effect); A2: adrenaline after thymoxamine (α-2 effect).</footnote>
<figure><img src="image_2.png" /><figcaption>FIG. 1. Effect of clonidine (0.1 µg/kg/min) on the electrical activity of the genital tract (integrated) in the absence of estradiol priming in an ovariectomized ewe. The effect was an enhancement of the occurrence of regular activity episodes at the cervix, without any evident effect on the uterus.</figcaption></figure>

abolished by prior administration of tolazoline or yohimbine.

Under the same conditions, infusion of phenylephrine or methoxamine remained without effect on both uterus and cervix.

2.) Study after estradiol priming. The i.m. administration of a single dose of 17β-estradiol elicited within 5-7 h, simultaneously, the outbreak of "spontaneous" activity at the uterus and the enhancement of the pre-existing activity of the cervix; both effects lasted for 96 to 120 h. Within this time, activity on both uterus and cervix was formed mainly by the alternation of 2 patterns, namely regular activity (as described above for the cervix), and irregular activity, formed by randomly occurring spikes that previously were shown to be related to phasic mechanical activity (Garcia-Villar et al., 1982). Twenty-four hours after estradiol administration and under beta blockade, the infusion of adrenaline triggered a sustained continuous activity on both uterus and cervix. At the cessation of the 20-min infusion, one or two well-individualized episodes of regular activity were observed; this corresponded to an overall increase of both irregular and regular activity patterns (Fig. 2A). This response was qualitatively modified by alpha blockers. Indeed, after administration of tolazoline or yohimbine, only continuous activity was recorded in response to adrenaline infusion, which corresponded to the reinforcement of irregular activity by α-1-receptor stimulation (Fig. 2B). In contrast, after thymoxamine or prazosin treatment, only a discrete response was recorded, corresponding to the enhancement of the regular activity pattern during and after adrenaline infusion (Fig. 2C). These two response patterns, i.e. continuous vs. discrete, were reproduced by phenylephrine and clonidine, respectively (Fig. 2D and 2E).

## Quantitative Study

Under estradiol priming, both uterus and cervix presented similar responsiveness to drugs. Therefore, the second part of our study was concerned with the

<figure><img src="image_3.png" /><figcaption>FIG. 2. Effects of α-1 and/or α-2 adrenoreceptor stimulation on cervix activity (integrated electromyograms) in one representative ewe under estradiol priming (P: propranolol). (A) Global response to α-1 and α-2 receptors simultaneous stimulation by adrenaline. (B, C) Specific responses to α-1 or α-2 receptors' selective stimulation by adrenaline after blockade by either tolazoline or thymoxamine. (D, E) Specific responses to α-1 or α-2 receptors' stimulation by relatively selective agonists i.e. phenylphrine or clonidine, respectively.</figcaption></figure>
quantitative evaluation of uterine responses to alpha-receptor stimulation. Adrenaline-infusion tests were carried out with and without α-1 (thymoxamine) or α-2 (tolazoline) blockers. Uterine activity responses were checked regularly after a single injection of 17β-estradiol (Time 0). The four response-time curves obtained for one complete series of infusions and for one representative ewe are shown in Figure 3. The highest activity levels were obtained during infusions of adrenaline alone (α-1 and α-2 simultaneous stimulation). Responses obtained for either α-1 (adrenaline infused after tolazoline) or α-2 (adrenaline infused after thymoxamine) specific stimulations were markedly lower. The development of the pharmacological response was analyzed mathematically. Several models based on different equations were tested: the best fit, as estimated by the Akaike's information criterion (Yamaoaka et al., 1978), was obtained with a set of three equations. Each equation describes a part of the curve according to time after estradiol injection (Fig. 4):

$$ (i) \text{ for } t < t_{\text{lag1}} \qquad E = E_0 \qquad \text{(Equation 1)} $$

$$ (ii) \text{ for } t_{\text{lag2}} < t < t_{\text{lag2}} \qquad E = E_0 + R_{\text{max}} [1 - e^{-k_1 (t - t_{\text{lag1}})}] \qquad \text{(Equation 2)} $$

$$ (iii) \text{ for } t > t_{\text{lag2}} \qquad E = E_0 + R_{\text{max}} [1 - e^{-k_1 (t - t_{\text{lag1}})}] e^{-k_2 (t - t_{\text{lag2}})} \qquad \text{(Equation 3)} $$

<figure><img src="image_4.png" /><figcaption>FIG. 3. Response-time curves plotted with data collected under the different experimental conditions of one complete series of infusions (see Table 1) in one representative ewe. Spontaneous activity (●-●) was measured during the hour that preceded each infusion of adrenaline alone (○-○-○); when relevant, adrenaline was infused after a single injection of tolazoline (■-■-■) or thymoxamine (□-□-□).</figcaption></figure>

where “t” was the time in hours; “t<sub>lag1</sub>” was the lag time between Time 0 and the first measurable uterine response, after “t<sub>lag1</sub>” uterine activity increased according to an apparent first order rate-constant “k1 (h⁻¹)”; “E” represented uterine response to adrenaline infusion and “Eo” was the spontaneous motility level in the absence of drug (in arbitrary computer unit); “t<sub>lag2</sub>” was the lag time between Time 0 and the onset of a process of decreasing responsiveness according to an apparent first order rate-constant “k2 (h⁻¹)”; “R<sub>max</sub>” was the theoretical maximum response when “t<sub>lag2</sub>”tended towards infinity.

With this model, an actual maximum response (E<sub>max</sub>) occurring at T<sub>max</sub> (Fig. 4) can be determined by successive iterations. Table 2 gives the average values of the equation parameters for controls and each type of treatment; E<sub>max</sub> values were significantly higher after adrenaline infusion than for controls or adrenaline infusion after thymoxamine (p<0.05, ANOVA, Newman-Keuls test); T<sub>max</sub> values were also significantly higher after adrenaline infusion than for adrenaline infusion after either thymoxamine or tolazoline. No significant differences were detected between the other parameters. From these data, it appeared that after a mean delay of 4.06 ± 0.78 h (t<sub>lag1</sub>) after estradiol administration, both spontaneous and drug-induced uterine activities began to increase, with an apparent half-time calculated from k<sub>1</sub> values) of 6.87 ± 3.64 h; thereafter, activity levels decreased, with an apparent half-time (calculated from k<sub>2</sub> values) of 12.54 ± 3.50 h.

<figure><img src="image_5.png" /><figcaption>FIG. 4. Theoretical response-time curve obtained with the proposed mathematical model (see text for definitions of parameters). Time 0: single injection of estradiol.</figcaption></figure>
<table><caption>TABLE 2. Parameters describing the development of uterine activity levels: values calculated from model equations.⁺</caption><thead><tr><th>Treatment</th><th>Eo (%)<sup>++</sup></th><th>E<sub>max</sub> (%)<sup>++</sup></th><th>t<sub>max</sub> (h)</th><th>kl (h<sup>-1</sup>)</th><th>t<sub>lag1</sub> (h)</th><th>k2 (h<sup>-1</sup>)</th><th>t<sub>lag2</sub> (h)</th></tr></thead><tbody><tr><td>Control</td><td>9.7 ± 8.7</td><td>54.6 ± 18.1<sup>a</sup></td><td>27.75 ± 21.00<sup>ab</sup></td><td>0.17 ± 0.16</td><td>4.54 ± 2.75</td><td>0.051 ± 0.049</td><td>26.16 ± 22.27</td></tr><tr><td>Adrenaline</td><td>15.6 ± 14.6</td><td>100 ± 0<sup>c</sup></td><td>36.00 ± 21.31<sup>b</sup></td><td>0.057 ± 0.068</td><td>3.42 ± 2.03</td><td>0.094 ± 0.10</td><td>31.70 ± 25.32</td></tr><tr><td>Adrenaline +tolazoline</td><td>13.0 ± 12.9</td><td>82.9 ± 12.4<sup>bc</sup></td><td>26.75 ± 13.33<sup>a</sup></td><td>0.11 ± 0.11</td><td>3.39 ± 2.76</td><td>0.049 ± 0.036</td><td>24.00 ± 16.82</td></tr><tr><td>Adrenaline +thymoxamine</td><td>11.5 ± 10.0</td><td>73.9 ± 18.5<sup>ab</sup></td><td>23.50 ± 12.87<sup>a</sup></td><td>0.14 ± 0.16</td><td>4.92 ± 1.87</td><td>0.046 ± 0.031</td><td>22.29 ± 13.70</td></tr></tbody></table>

DISCUSSION

The in vivo pharmacological approach used in the present experiments provided the following information. First, both alpha-adrenoreceptor subtypes were present in the ewe genital tract (uterus and cervix). Second, their selective activation by means of appropriate agonists and antagonists led to increases in uterine and cervical activities. These effects were under estrogenic control (uterus) and/or regulation (cervix): the cervix being able to contract in response to α-2-receptor stimulation in the absence of estrogen priming. Thirdly, under estradiol priming, responses to either α-1- or α-2-receptor activation differed qualitatively: each type enhancing selectively one of the two activity patterns previously described for the ovine genital tract (Garcia-Villar et al., 1982). Indeed, utero-cervical responses to α-1-receptor activation took the form of continuous irregular activity, whereas responses to α-2-receptor activation took the form of discrete episodes of regular activity.

These qualitatively different effects support the hypothesis that the physiological occurrence of genital tract contractions may be under the control of two separate and, according to the hormonal status, complementary systems. A first system, subserved by α-2 receptors, is responsible for the frequency of a basic rhythm constituted by the cyclic recurrence of activity episodes. This chronotropic function of uterine α-2 receptors could be exerted via the activation of a hypothetical trigger zone operating at the cervix (as shown in the present study) and at the small curvature of uterine horns (unpublished observation), even in the absence of estrogen priming. In contrast, estrogen priming would be needed for the α-2 receptors to operate on the major part of the uterus. On the other hand, a second system, subserved by α-1 receptors, would regulate the magnitude

of the irregular activity (i.e. frequency and force of phasic contractions) that appeared on the whole genital tract only under estrogen priming. This would tentatively qualify as an ionotropic function of uterine α-1 receptors, since our electrical recordings have been shown to correlate well with muscle contractility.

This new hypothesis concerning chronotropic and inotropic functions for α-2 and α-1 uterine adreno-receptors, respectively, invites speculation concerning the action of drugs on the uterine tract. These drugs are generally classed as tocolytic (inhibitory) or oxytocic (excitatory). Now, well-defined activity patterns comparable to those described for sheep have been reported in several species (e.g. Ruckebusch and Bayard, 1975; Taverne et al., 1979; Scheerboom et al., 1986). Our study revealed that drugs can interfere with both, or with one of the basic patterns of activity. Consequently, we suggest that this qualitative aspect be taken into account when the effects of oxytocic drugs are described. In this respect, it must be recalled that, since Szurszewski (1969) described the migrating motor complex of the in-testine (MMC), drug effects on gastrointestinal motility are qualified by the authors in this field, mainly in terms of disruption or enhancement of the basic MMC pattern.

An in vivo pharmacokinetic approach was used to analyze, according to a set of three equations, the development of spontaneous and drug-induced uterine motility after a single administration of estradiol. The first objective of our analysis was to express concisely a relatively complex curve. Based upon a limited number of parameters, an objective and accurate comparison was obtained between the four response-time curves. The second objective was to represent the development, with time, of the

<footnote>⁺Values are means ± SD (n = 8); different superscripts indicate significant differences (p<0.05, ANOVA and Newman-Keuls test).</footnote>

<footnote>⁺⁺Percentage of E<sub>max</sub> obtained during adrenaline infusion test.</footnote>
actual biological mechanisms underlying the response. Our three equations were not only analytical expressions but were constructed from the following physiological hypotheses: 1.) the pharmacological response must be related in some way to the presence of specific alpha receptors; 2.) after estradiol administration, a lag-time was necessary for the synthesis of new receptors; and 3.) receptors appeared and disappeared according to first order kinetics, the disappearance of receptors beginning after a second lag-time. These hypotheses were supported by in vitro investigations. Indeed, in the rabbit, an estrogen-induced increase in myometrial alpha-adrenergic-binding sites occurred in a time-dependent and dose-dependent manner and alpha-adrenergic-receptor concentration did not increase until more than 6 h after the injection of estrogen (Roberts et al., 1981). This delay is comparable to the lag-time ($t_{lag}$) reported in the present experiment. The same authors also reported that receptor concentration was near maximal after a delay of 12 h post-estradiol treatment, the number of binding sites returning to preinjection levels by the 4th day. In the present study, after a delay of 12 h the calculated response was 86.55% of the maximum response and about 13.62% at 96 h after estradiol administration (Table 3). Owing to the similarity between this in vitro and our in vivo studies, it seems reasonable to assume that kinetics of effects would be related to kinetics of alpha receptors. If this assumption is valid, then two conclusions can be drawn. First, the time course of appearance and disappearance was similar for $\alpha$-1 and $\alpha$-2 estrogen-induced adrenoreceptors. Second, since this kinetic paralleled that of spontaneous

uterine motility, it could be suggested that, under physiological conditions, alternate activation by sympathetic nerves of either type of alpha receptor may be responsible for the alternation of the irregular and regular activities previously described for the estrogen-primed uterus (Garcia-Villar et al., 1982).

Nevertheless, the major weakness of these hypotheses was that our pharmacological approach reliably defines the types of receptors and their relative abundance only if factors acting between receptor and effector are not altered by other mechanisms. One of these could be the sensitization of the myometrium by estrogen. Indeed, according to Rexroad (1981) the number and/or affinity of uterine alpha receptors would not be enhanced by estrogen. In this case, our kinetic results mainly would reflect the time course of appearance and disappearance of gap junctions after estrogen priming (Garfield et al., 1979; Verhoeff et al., 1985).

## ACKNOWLEDGMENTS

Our thanks are due to Mr J. P. Soula, R. Theisen, and Mme C. Soula for their excellent care of the animals and to C. Santamaria for typing the manuscript.

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<table><caption>TABLE 3. Theoretical effects for Times 0, 12, and 96 h after estradiol injection, calculated according to the model proposed.</caption><thead><tr><th>Treatment</th><th>Eo<sup>+</sup></th><th>E<sub>12</sub></th><th>E<sub>max</sub><sup>++</sup></th><th>E<sub>96</sub></th></tr></thead><tbody><tr><td>Control</td><td>11.2<sup>+++</sup></td><td>96.7</td><td>100</td><td>14.6</td></tr><tr><td>Adrenaline</td><td>9.4</td><td>63.9</td><td>100</td><td>9.7</td></tr><tr><td>Adrenaline + tolazoline</td><td>10.5</td><td>91.5</td><td>100</td><td>14.4</td></tr><tr><td>Adrenaline + thymoxamine</td><td>11.4</td><td>94.1</td><td>100</td><td>15.8</td></tr><tr><td>Overall means</td><td>10.62</td><td>86.55</td><td>100</td><td>13.62</td></tr></tbody></table>

+Eo was calculated by equation 1, $E_{12}$ by equation 2, $E_{96}$ by Equation 3 (see text).

++Emax was calculated by successive iterations using Equation 3.

+++Values are means (n = 8) expressed in percent of Emax.
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