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

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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

To cite this version:

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, Society for the Study of Reproduction, 1987, 37 (1), pp.241-248. �10.1095/bi- olreprod37.1.241�. �hal-02719421�

In Vivo Pharmacological Characterization of Alpha Adrenergic Receptors in Sheep Myometrium and Their Physiological Meaning’

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-i adrenergic mechanisms. Alpha-2 receptors could be assigned

to a chronotropic function by activating a hypothetical zone triggering the outbreak of discrete episodes of mo-

tility (regular activity); this function is apparent at the cervix even in the absence of estrogen priming. On the

other hand, alpha-i receptors could be assigned to an inotropic function, regulating mainly the magnitude of

estrogen-dependent utero-cervical motility (irregular activity).

241 INTRODUCTION

In several species, including sheep, the uterus contains both alpha (a) and beta (3) adrenoreceptors mediating, respectively, excitatory and inhibitory responses to catecholamines (Alhquist, 1948; Mar- shall, 1970). Pharmacological properties and physio- logical significance of uterine beta receptors are well known due to extensive therapeutic use of j3-2 ag- onists. In contrast, corresponding information con- cerning alpha receptors is rather scarce. By studying

the displacement of 3H-dihydroergocriptine from

rabbit uterine cell membrane by prazosin and yohim- bine, it was clearly demonstrated that both subtypes of adrenoreceptors were present in the rabbit uterus (Hoffman et al., 1981). In some other species, pre- sence of both a-i and a-2 adrenoreceptors is probable.

In the cyclic sow, it was shown that most of the

uterine alpha receptors were of the a-2 subtype

(Rexroad and Guthrie, 1983). In sheep, uterine alpha receptors display a lower affinity for phenylephrine, a

Accepted February 16, 1987.

Received July 28, 1986.

‘This work was taken from a chapter of Dr. P. G. Marnet’s Doctoral

Thesis.

2Reprint requests: Dr. R. Carcia-Villar, Station de Pharmacologic-

Toxicologic, I.N.R.A., 180 chemin de Tournefeuille, 31300 Toulouse,

France.

potent a-i agonist, than for epinephrine and nor- epinephrine, which share both a-i and a-2 potencies

(Rexroad, 1981), suggesting that a-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 a-2 receptors (Hoffman et al., 1981). Conversely, proges- terone treatment of ovariectomized ewes increased

the concentration of alpha receptors (Rexroad,

1981), which were probably of the a-2 subtype.

Similarly, in the pig, a-2 receptors were more numer- ous 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 a-i receptors were coupled to contractile responses, whereas a-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 a-i receptors.

These findings contrasted with those reported in the same species by Roberts et a!. (1981) who concluded that estrogen increased both populations of alpha receptors and responsiveness of the uterus.

242 ^{MARNET ET AL.}

In intact cyclic or ovariectomized estradiol-treated ewes, phenylephrine or methoxamine, which display a selective affinity for a-i 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 pre-

dominantly an a-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 a-i or a-2 agonists were not qualitatively equivalent, each type of agonist leading to the pre- ferential 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 (a-2 agonists) and irregular activity (a-i agonists). The objectives of the present work were to qualify the nature of the a-i vs. a-2 adreno- receptor-induced uterine activations and their control by estrogens, and to suggest a hypothesis concerning their possible involvement in the regulation of phys- iological 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 intra- parietal 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 a!., 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 auto- matically 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 a-i and a-2 potencies) was infused after beta receptor blockade to test the overall response to alpha receptor simulation; a-l-

and a-2-receptor-mediated responses were then

demonstrated by the use of selective a-2 and a-i

antagonists, respectively. Adrenaline was infused for 20 mm (1 i.tg/kg/min) in the jugular vein by means of a portable syringe driver (MS 16, Graseby Medical, UK) through an indwelling catheter. All infusions started 10 mm after a single i.v. injection of pro- pranolol; the dosage of propanolol 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 a-i mediated effect of adrenaline was evi-

denced after yohimbine (1 mg/kg) or tolazoline

(1 mg/kg) administration. Under the same conditions, a-2-mediated effects of adrenaline were evidenced after administration of prazosin (1 mg/kg) or thymo- xamine (1 mg/kg). Antagonists were administered by

venipuncture in the opposite jugular vein 5 mm

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-mm infusions of specific a-i agonists, i.e., phenylephrine (5 ,ug/kg/min) or methoxamine (0.4 mg/kg/mm), and a-2 agonists, i.e. clonidine (0.3 pg/kg/mm) or xylazine (1 pg/kg/mm), 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 i713-estradiol (2 pg/kg in

ethanol). Adrenaline infusions were performed, as

described above, before and regularly after estrogen priming. Thymoxamine and tolazoline, respectively, were selected as a-i and a-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 1. General schedule of drug administration for the quantitative study.

Time a ccording to estrogen (h) First experiment series Second experiment series

(1) (2) (3) (1) (2) (3)

(1) (2) (3)

-24 -20 -16 A2 Al A Al A2

2.5 3.5 - A Al - A A2 -

5 6 - A A2 - ^{A Al} -

7.5 8.5 - ^{A Al - A A2} -

10 11 - A A2 - A Al -

24 28 32 A Al A2 A A2 Al

48 52 56 A A2 Al A Al A2

72 76 80 A Al A2 A A2 Al

96 100 104 A A2 Al A Al A2

120 124 128 A Al A2 A A2 Al

168 172 176 A A2 Al A Al A2

216 220 224 A Al A2 A A2 Al

+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.

++A: Adrenaline alone (overall a effect);Al: adrenaline after tolazoline (a-i effect); A2: adrenaline after thymoxamine (a-2 effect).

activity was quantified every 20 sec by the on-line

microcomputer. Mean values were automatically

calculated for periods of 5 mm 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 mm of infusion.

For each animal and for each of the two periods, the highest observed response to the adrenaline test was

recorded as iOO% 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 (Koeppe and Hamann, 1980).

Initial estimates were obtained using linear regression methods.

Drugs

The drugs used were adrenaline (ADRENALINE,

Aguettant, Lyon, France); propranolol (AVLO-

CARDYL, IC! Pharma, Enghien, France); yohimbine hydrochloride (Sigma Chemical Co., St. Louis, MO);

tolazoline hydrochloride (Sigma Chemical Co.);

prazosin hydrochloride (Pfizer Inc. Orsay, France);

thymoxammne (MOXISYLYTE, Dedieu, Bordeaux,

France); phenylephrmne (NEOSYNEPHR!NE, Boehrin-

ger !ngelheim, Reims, France); methoxamine (VASO- XYL, Wellcome, London, England); clonidine (CATA-

PRESSAN, Boehringer Ingelheim); xylazine (ROM-

PUN, Bayer, Leverkussen, G. F. R); dimethylsuiph-

oxyde (Farmitalia Carlo-Erba, Milan, Italy); and

173-estradiol (Sigma Chemical Co.).

Qualitative Study

RESULTS

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-mm 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-mm period between 2 episodes of regular activity and after the blockade of beta recep- tors by propanolol, a motor response restricted to the cervix was observed. It consisted of an episode of electrical activity lasting 12.91 ± 3.38 mm (mean ±

SD; n ⁼ 23), ending always before the 20-mm in-

fusion was completed. This cervical response was

blocked by prior administration of either tolazoline or yohimbine, but not by thymoxamine or prazosin.

Similarly, a 20-mm 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

UTERUS

(B)

abolished by prior administration of tolazoline or Quantitative Study

yohimbmne. Under estradiol priming, both uterus and cervix

Under the same conditions, infusion of phenyl- presented similar responsiveness to drugs. Therefore,

ephrine or methoxamine remained without effect on the second part of our study was concerned with the

both uterus and cervix.

2.) Study after estradiol priming. The i.m. admin- istration of a single dose of i713-estradiol elicited

within 5-7 h, simultaneously, the outbreak of

“spontaneous” activity at the uterus and the en-

hancement of the pre-existing activity of the cervix;

both effects lasted for 96 to 120 h. Within this time,

.

actlvlty on both uterus and cervix was formed mainly

i iii

I

A

ADRENALINE IOn,In

P

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 a!., 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-mm 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 administra- tion of tolazoline or yohimbine, only continuous activity was recorded in response to adrenaline infusion, which corresponded to the reinforcement of irregular activity by a-i-receptor stimulation (Fig.

2B). In contrast, after thymoxammne 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).

0 mu,

(C)

2mv

ADRENALINE THY MOX AMINE

0mm

(D

I2mui

PH EN YLEPH PINE

10 ‘nun

(E) I

ILlJLiJ

iJ14 ^1j1

CLONIDI NE ^{0 nfl}

FIG. 2. Effects of a-l and/or a-2 adrenoreceptor stimulation on

cervix activity (integrated electromyograms) in one representative

ewe under estradiol priming (Pu propranolol). (A) Global response to

a-l and a-2 receptors simultaneous stimulation by adrenaline. (B, C)

Specific responses to a-i or a-2 receptors’ selective stimulation by

adrenaline after blockade by either tolazoline or thymoxamine. (D, E)

Specific responses to cs-I or a-2 receptors’ stimulation by relatively selective agonists i.e. phenylphrine or clonidine, respectively.

244 ^{MARNET ET AL.}

2 my

- i - . *

I

CERVIX

Ai JL

CLONIDINE 0mm

FIG. 1. Effect of clonidine (0.1 zg/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.

ADRENALINE

PL+ TOLAZOLINE

75

>- I.-

>

134

I- U U.’

-J UJ

0

-24 #{212} +24

‘ k2

72 20 68 216

HOURS AFTER ESTRADIOL

.-.CONTROt. 0--a p,fl .- +Q(_ O--QADRsTXM

(man

(of _________

4______________

r^tiog2

0

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 (o- -^{-0); when relevant, adrenaline was infused after a single}

injection of tolazoline (.-.) or thymoxamine (o- -^-a).

50 100 ISO

HOURS AFTER ESTRADIOL

FIG. 4. Theoretical response-time curve obtained with the proposed

mathematical model (see text for definitions of parameters). Time 0:

single injection of estradiol.

quantitative evaluation of uterine responses to

alpha-receptor stimulation. Adrenaline-infusion tests were carried out with and without a-i (thymoxamine) or a-2 (tolazoline) blockers. Uterine activity res- ponses were checked regularly after a single injection of 1713-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 (a-i and a-2 simUl- taneous stimulation). Responses obtained for either

a-i (adrenaline infused after tolazoline) or a-2

(adrenaline infused after thymoxamine) specific

stimulations were markedly lower. The development of the pharmacological response was analyzed math- ematically. Several models based on different equa- tions were tested: the best fit, as estimated by the Akaike’s infomation criterion (Yamaoka et a!., 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)fort< t1

E ⁼ Eo (Equation 1)

(ii) for tlag2<t<tlag2

E ⁼ Eo + Rm 1ie1(ttiagi)] (Equation 2)

(iii) for t> t2

E ⁼ Eo + Rm [1e_k1(ttag1)]ek2(tt1ag2)

(Equation 3)

where “t” was the time in hours; “t1” was the lag

time between Time 0 and the first measurable uterine response, after “tj1” uterine activity increased according to an apparent first order rate-constant “kl (h )“; “E” represented uterine response to adren- aline infusion and “Eo” was the spontaneous motility level in the absence of drug (in arbitrary computer unit); “tlag2” 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’ )“; “Rmax” was the theoretical maximum

response when “tlag2 “tended towards infinity.

With this model, an actual maximum response

(Em ax) occurring at Tmax (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; Emax values were signifi-

candy higher after adrenaline infusion than for

controls or adrenaline infusion after thymoxamine

(p<0.05, ANOVA, Newman-Keuls test); ^Tmax values

were also significantly higher after adrenaline infusion than for adrenaline infusion after either thymo- xamine 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 (ti) after estradiol administration, both spontaneous and drug-induced uterine activities began to increase, with an apparent half-time cal- culated from k1 values) of 6.87 ± 3.64 h; thereafter, activity levels decreased, with an apparent half-time (calculated from k2 values) of 12.54 ± 3.50 h.