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Effect of progesterone on adrenoceptors in the isthmus of the rabbit oviduct
European Journal of Pharmacology, 48 (1978) 403--410 © Elsevier/North-Holland Biomedical Press
403
E F F E C T OF P R O G E S T E R O N E ON A D R E N O C E P T O R S IN THE I S T H M U S OF THE R A B B I T OVIDUCT DORIANNE E. RHEAUME and DAVID M. PATON * Department of Pharmacology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada
Received 20 December 1977, accepted 2 January 1978
D.E. RHEAUME and D.M. PATON, Effect of progesterone on adrenoceptors in the isthmus of the rabbit oviduct, European J. Pharmacol. 48 (1978) 403--410. The effect of adrenergic agonists on the contractility of circular muscle of the isthmus of the oviduct was studied in isolated tissues from rabbits in estrus and after progesterone pretreatment, a-Adrenoceptor sensitivity was not altered by progesterone, and the order of potencies of a-agonists was similar to that for a-adrenoceptors in other rabbit tissues. Progesterone pretreatment increased the maximal inhibitory effect of isoprenaline on ~-adrenoceptors, but did not alter sensitivity to the agonist, suggesting that there had been an increase in the number of ~-adrenoceptors rather than a change in their affinity. The order of potencies of ~-agonists was similar to that for the ~l-adrenoceptor in other rabbit tissues.
Rabbit oviduct
Adrenoceptors
Estrus
1. I n t r o d u c t i o n T h e p r e s e n c e o f a - e x c i t a t o r y a n d ~-inhibit o r y a d r e n o c e p t r o s in r a b b i t o v i d u c t is well e s t a b l i s h e d ( L o n g l e y et al., 1 9 6 8 ; Martin et al., 1970; Levy and Lindner, 1972; Hodgson and Pauerstein, 1974, 1975). a-Adrenoceptor a c t i v i t y p r e d o m i n a t e s u n d e r all h o r m o n a l cond i t i o n s since a d r e n a l i n e a n d n o r a d r e n a l i n e a l w a y s elicit c o n t r a c t i l e r e s p o n s e s t h a t are blocked by phentolamine or phenoxybenzamine. N o r a d r e n a l i n e h a d a d e c r e a s e d p o t e n c y on t h e circular m u s c l e o f t h e i s t h m u s o f r a b b i t o v i d u c t in p r o g e s t e r o n e - d o m i n a n t as c o m p a r e d to e s t r o u s tissues (Higgs a n d M o a w a d , 1974; Rheaume and Paton, 1977a). Inhibition o f c a t e c h o l a m i n e u p t a k e processes did n o t * Present address and to whom correspondence should be addressed: Department of Pharmacology and Clinical Pharmacology, School of Medicine, University of Auckland, Auckland, New Zealand.
Isoprenaline
Progesterone
Noradrenaline
e l i m i n a t e t h e s e p o t e n c y d i f f e r e n c e s since, in t h e p r e s e n c e o f cocaine, h y d r o c o r t i s o n e or b o t h agents, n o r a d r e n a l i n e was sill less p o t e n t in p r o g e s t e r o n e - d o m i n a n t tissues ( R h e a u m e a n d P a t o n , 1 9 7 7 a ) . A r e d u c t i o n in a - a d r e n o c e p t o r s a c t i v i t y ( H u n t e r a n d Kendle, 1 9 7 4 ; H e i l m a n et al., 1 9 7 6 ) or, c o n v e r s e l y , an increase in ~ - a d r e n o c e p t o r a c t i v i t y (Martin et al., 1 9 7 0 ; H o d g s o n a n d Pauerstein, 1 9 7 4 , 1 9 7 5 ) have b e e n p r o p o s e d to e x p l a i n e f f e c t s o f p r o g e s t e r o n e o n r a b b i t o v i d u c t a l isthmus. Such changes in t h e relative sensitivity o f t h e t w o a d r e n o c e p t o r s w i t h changes in n o r m a l d o m i n a n c e ma3 b e o f p h y s i o l o g i c a l signific a n c e since a d m i n i s t r a t i o n o f p r o g e s t e r o n e before ovulation accelerated ovum transport in t h e r a b b i t ( P a u e r s t e i n et al., 1 9 7 4 ) . In t h e p r e s e n t s t u d y we have investigated t h e e f f e c t o f adrenergic agonists o n t h e cont r a c t i l i t y c,,f isthmic circular m u s c l e o f r a b b i t s in estrus a n d a f t e r a p r o g e s t e r o n e - p r e t r e a t m e n t schedule k n o w n to a c c e l e r a t e o v u m t r a n s p o r t ( P a u e r s t e i n et al., 1 9 7 4 ) a n d have studied t h e
404 effects of these hormonal states on a- and fladrenoceptor activity, a- and fi-adrenoceptors in oviductal isthmus were also characterized by determining the order of potency of adrenergic agonists on each receptor system. A preliminary account of this research has been published (Rheaume and Paton, 1977b).
2. Materials and methods
2.1. Animals and hormone pretreatment Mature, female New Zealand white rabbits (3--5 kg) were emplyed. Two hormone groups were studied: Group I, Estrus received 17/3estradiol (5 pg, s.c. daily) for 6 days; Group II, Human Chrionic Gonadotropin (HCG)+ Progesterone Pretreatment received progesterone (2.5 mg, i.m.) on the day of HCG injection (100 iu, i.v.) and on each of the two preceding days. This treatment accelerated ovum transport in the rabbit (Pauerstein et al., 1974). Rabbits in Group II were killed 20 h after HCG administration.
2.2. Measurement o f contractile responses Rabbits were killed by cervical dislocation. After the oviducts had been dissected free of surrounding tissue, the isthmus was identified and cut into 2--3 mm pieces. Oviductal segments were threaded twice through the lumen, and attached at the b o t t o m of the tissue bath and to a transducer. These circular muscle preparations were set up under 0.5 g resting tension in baths containing modified Krebs solution at 28°C, pH 7.4 and bubbled with 95% 02--5% CO2. Tissues were allowed to equilibrate for at least 45 min before any experimental procedure was begun. The modified Krebs solution had the following composition (mM): NaC1 116, KC1 5; CaC12 1.25; MgC12 1.2; NaH2PO4 1.2; NaHCO3 22; D-glucose 11.2. Ascorbic acid, 0.1 mM and Na2EDTA, 0 . 0 4 m M were added to prevent oxidation of amines. Responses to adrenergic agonists were
D.E. RHEAUME, D.M. PATON measured as changes in isometric tension with Grass (FT.03C) Force Displacement Transducers and displayed on a Grass (Model 5D) Polygraph. Responses were allowed to reach a maximum before the drugs were washed out.
2.3. Determination o f hormonal status Uterine histology was examined to define hormonal status, using Johnson's Method for Metachromasia (0.1% toluidine blue) (Luna, 1968). In the Estrous group the endometrium exhibited a proliferative phase; HCG + Progesterone-pretreated endometrium exhibited a secretory phase.
2.4. Dose--response determinations and EDso calculations Non-cumulative concentration-response curves to (--)-adrenaline, (--)-noradrenaline and (--)-phenylephrine were determined using at least 5 animals for each study. Responses were expressed as a percentage of the maximal responses of the tissue (to 10 -4 M adrenaline). Potencies of adrenergic agonists were compared in terms of EDs0 values, which were extrapolated by computer regression analysis of values between 20--80% on the log concentration/response curves. Relative potencies of agonists were expressed as the ratio of EDs0 values, taking the potency of (--)-noradrenaline as 1.0. The effects of various agents on t h e potencies of the adrenergic agonists were examined in dose--response determinations performed after a 20 min exposure to the drug. All drugs were present during the dose--response determination and comparison was made with a control tissue in each experiment.
2.5. a-Adrenoceptor activity The order of potency of adrenergic agonists on ~-adrenoceptors was also determined. Catecholamine uptake processes were blocked with cocaine (3 X 10 -s M) and hydrocortisone (10 -4 M), and t3-adrenoceptors were blocked with propranolol (3.8 X 10 -6 M).
PROGESTERONE AND ADRENOCEPTORS IN OVIDUCT
405
2.6. Characterization of [J-adrenoceptors
2.7. Drugs and chemicals
2.6.1. {J-Adrenoceptor activity in estrous and HCG + progesterone pretreated isthmus fl-Adrenoceptor activity was determined by examining the (+)-isoprenaline antagonism of acetylcholine contractile responses using a modification of the method described b y Hodgson and Pauerstein (1975). At concentrations greater than 10-SM, (+)-isoprenaline exhibits a-excitatory activity in this tissue. Tissue were therefore exposed to phenoxybenzamine (10 -6 M) for 20 min to block aadrenoceptors. Cocaine (3 X 10 -s M) and hydrocortisone (10-4M) were included to block neuronal and extraneuronal uptake of catecholamines. Contractile responses to a maximal dose of acetylcholine (5 X 10 -4 M) were elicited every 15 min. Then, 2 min before the addition of acetylcholine, (+)-isoprenaline (2 X 10 -7 to 10 -4 M) was administered. The % inhibition of the acetylcholine response was calculated b y comparing the magnitude of the response in the presence of (+)-isoprenaline to the mean of control responses before and after. The maximum inhibition of response as well as IDs0 values were compared between hormone treatment groups.
The following drugs were used in this study: (+)-salbutamol sulphate * (Allen & Hanburys, Ltd.}; p r a c t o l o l * and propranolol hydrochloride * (Ayerst, McKenna & Harrison, Ltd.); cocaine hydrochlride (British Drug House, Ltd.), 1713-estradiol (Calbiochem); papaverine hydrochloride (Eli Lilly & Co.); acetylcholine hydrochloride, (--)-adrenaline bitartrate, human chorionic gonadotropin, hydrocortisone 21-sodium succinate, (+)isoprenaline hydrochloride, (--)-phenylephrine hydrochloride, progesterone (Sigma Chemical Co.), phenoxybenzamine hydrochloride * (Smith, Kline & French, Ltd.). Compounds indicated with an asterisk were generously donated by the companies listed.
2.6.2. Order of agonist potency on [J. adrenoceptors A series of experiments as described above was completed for the antagonism by (--)adrenaline, (--)-noradrenaline and (+)-salbutamol of the acetylcholine response in Estrous and HCG + Progesterone-pretreated tissues. The order of agonist potency on fl-adrenoceptors was ascertained and the maximum inhibitory effects of the agonists noted. 2.6.3. Non-specific inhibition of the acetylcholine response Dose--response curves for papaverine inhibi. tion of the cholinergic contractile response were similarly studied in the two hormone groups. The IDs0 values and maximum inhibitory responses were compared between groups.
2.8. Statistical analysis Resulting were expressed as the mean + S.E.M. Student's unpaired two-tailed t-test was employed to make comparative assessments. 95% was chosen as an acceptable level of significance.
3. Results
Responses of tissues were studied at 28°C and in the presence of a reduced concentration of Ca 2÷ (1.25 mM) because spontaneous activity was considerably reduced under these conditions, and responses to agonists and to transmural stimulation did not display desensitization with time. At 37°C there was a timedependent reduction in responses to both (--)-noradrenaline and to transmural stimulation, possibly due to prostaglandin liberation as the reduced responsiveness was abolished by indomethacin (Paton and Johns, 1975). 1
3.1. a-Adrenoceptor sensitivity a-Adrenoceptor sensitivity did not vary between groups since the potency of (--)phenylephrine in the presence of propranolol
406
D.E. R H E A U M E , D.M. P A T O N A
B
3.2. Characteristics of [J-adrenoceptors
10
10
Fig. 1. a - A d r e n o c e p t o r sensitivity of rabbit oviductal isthmus in Estrous (open bar) and HCG + Progesterone-pretreated (hatched bar) tissues. The histogram represents the EDs0 for: (A) (--)-phenylephrine (in the presence of 3.8 x 10 .6 M propranolol); and (B) for (--)-adrenaline (in the presence of 3 × 10 -s M cocaine, 10 -4 M h y d r o c o r t i s o n e and 3.8 × 10 .6 M propranolol). Mean ± S.E.M. of 5--6 observations. Ordinate: (A) EDs0 (×10 -6 M); (B) EDs0 (x 10 -7 M).
(3.8 X10-6 M) and the potency of (--)adrenaline in the presence of c o c a i n e + hydrocortisone + propranolol were not altered by either of the hormonal treatments (fig. 1). The order of p o t e n c y for the adrenergid agonists on a-adrenoceptors (i.e., in the presence of cocaine + hydrocortisone + propranolol) was determined to be (--)-adrenaline > (--)-noradrenaline > (--)-phenylephrine, the EDs0 values being 5.0 X 10 -7 M, 6.8 X 10 -7 M and 3.8 X 10 .6 M respectively. In table 1 the relative potencies for the adrenergic agonists on a-adrenoceptors in oviductal isthmus are compared with values for other rabbit tissues.
3.2.1. ~-Adrenoceptor activity in Estrous and HCG ÷ Progesterone-pretreated isthmus (+)-Isoprenaline antagonism of the acetylcholine-elicited contractile response was studied to provide a measure of fi-adrenoceptor activity in Estrous and HCG + Progesteronepretreated isthmus. In progesterone-pretreated tissues, the ~-agonist maximally inhibited the contractile response to acetylcholine by about 90%, while in Estrous tissues, (+)-isoprenaline only maximally inhibited the acetylcholine response by about 30% (fig. 2). Propranolol (3.8 X 1 0 -6 M) blocked the effect of (+-)-isoprenaline in both groups. The absolute magnitude of the contractile response to acetylcholine (5 X 10 -4 M) was significantly greater in Estrous tissues (0.42 + 0.05 g, n = 7) than in HCG + Progesteronepretreated tissues (0.27 + 0.03 g, n = 13). In spite of this, the absolute magnitude of the inhibition of the acetylcholine response produced by (+)-isoprenaline was still greater in the HCG + Progresterone group (0.24 g) than in the Estrous group (0.13 g). Normalized curves for the inhibitory effect of (+)-isoprenaline were also determined by expressing each inhibitory response as a % of the maximum inhibitory response for each treatment group, and b y plotting against (+-)isoprenaline concentration (fig. 3). When expressed in this manner, the IDs0 values were not found to differ between the Estrous
TABLE 1 Relative potencies of adrenergic agonists on a-adrenoceptors in various rabbit tissues. Tissue
r. Thoracm aorta 1 Duodenum 1 Stomach fundus 1 Oviductal isthmus (estrus)
1 F r o m F u r c h g o t t , 1972.
Relative potencies (--)-Adrenaline
(--)-Phenylephrine
(--)-Noradrenaline
1.2 2.0 1.0 1.4
0.2 0.25 0.25 0.2
1 1 1 1
P R O G E S T E R O N E A N D A D R E N O C E P T O R S IN O V I D U C T
407
I00
I00
80
80
60
60 40
40
20
2O
0 0
I
I
I
10-6
I0-~
10-4
I
2 xlO-7
L 2xl()-7
~-6
~[s
~-4
Fig. 2. ~ - A d r e n o c e p t o r activity o f r a b b i t o v i d u c t a l i s t h m u s in E s t r o u s a n d HCG + P r o g e s t e r o n e - p r e t r e a t e d i s t h m u s . Each p o i n t r e p r e s e n t s t h e m e a n ± S.E.M. o f 6--8 o b s e r v a t i o n s o f t h e % i n h i b i t i o n o f t h e a c e t y l c h o l i n e c o n t r a c t i l e r e s p o n s e b y (±)-isoprenaline. E s t r o u s group, o o; HCG + Progesterone, • =. O r d i n a t e : % i n h i b i t i o n o f r e s p o n s e t o a c e t y l c h o l i n e (5 x 10 -4 M). Abscissa: i s o p r e n a l i n e c o n c e n t r a t i o n (M).
Fig. 3. N o r m a l i z e d curves for t h e i n h i b i t o r y e f f e c t o f (+-)-isoprenaline o n t h e a c e t y l c h o l i n e r e s p o n s e in Est r o u s a n d H C G + P r o g e s t e r o n e - p r e t r e a t e d tissues. Each i n h i b i t o r y r e s p o n s e is e x p r e s s e d as a % o f t h e m a x i m u m i n h i b i t o r y r e s p o n s e for t h a t h o r m o n e g r o u p -+S.E. E s t r o u s group, o o; H C G + P r o gesterone, • =. 6--8 o b s e r v a t i o n s . O r d i n a t e : % m a x i m u m i n h i b i t o r y r e s p o n s e ; abscissa: i s o p r e n a l i n e c o n c e n t r a t i o n (M).
and HCG + Progesterone groups being 3.4 X 10 -7 M and 5.2 X 10 -7 M respectively. Thus, while the maximum inhibitory effect was greater in the HCG + Progesterone group, the potency of (+)-isoprenaline on ~-adrenoceptors did not vary.
ceptors in HCG + Progesterone-pretreated tissues was (+ )-isoprenaline /> (--)-noradrenaline > (--)-adrenaline ~ (+)-salbutamol, the EDs0 values being 5.1 X 10 -TM, 5.2 × 10 -TM, 1.3 X 1 0 -6 M a n d > 1 0 -4 M respectively. Table 2 lists the potencies for the adrenergic agonists on fi-adrenoceptors in oviductal isthmus along with comparable values for other rabbit tissues. (--)-Adrenaline and (--}-noradrenaline produced maximum inhibitory
3.2.2. Order o f agonist potency on fiadrenocep tors The order of agonist potency on 13-adreno-
TABLE 2 Relative p o t e n c i e s of a d r e n e r g i c agonists o n ~3-adrenoceptors in various r a b b i t tissues. Tissue
Thoracic aorta 1 Left atrium 1 Small i n t e s t i n e 1 Stomach fundus I Oviductal isthmus (HCG + P) 1 F r o m F u r c h g o t t , 1972.
Relative p o t e n c i e s (--)-Isoprenaline
(--)-Adrenaline
(--)-Noradrenaline
(+)-Salbutamol
130 3.5 1.5 2.5 1.0
65 0.5 0.2 1.2 0.4
1 1 1 1 1
< 0.01
408
D.E. R H E A U M E , D.M. P A T O N
10C
8C
60
4C
i
3 × 1 0 -'s
l
10-5
I
3 x 1 0 -5
I
10 -4
i
3 x 1 0 -3
Fig. 4. I n h i b i t o r y e f f e c t of p a p a v e r i n e o n t h e acetylc h o l i n e response in E s t r o u s a n d H C G + P r o g e s t e r o n e p r e t r e a t e d r a b b i t isthmus. Each p o i n t r e p r e s e n t s the m e a n % i n h i b i t i o n _+S.E. o f 5 observations. Estrus, o o; H C G + P r o g e s t e r o n e , • ~. O r d i n a t e : % i n h i b i t i o n o f r e s p o n s e t o a c e t y l c h o l i n e (M); abscissa: p a p a v e r i n e c o n c e n t r a t i o n (M).
effects of the same order of magnitude as (+)isoprenaline. However, (+)-salbutamol, a specific ~2-agonist, failed to achieve such a maximum effect, and at 1 0 - 4 M elicited only 40--50% of the maximum inhibitory response to (+)-isoprenaline.
3.2.3. Non-specific inhibition o f the acetylcholine response Papaverine exhibited identical dose-response curves in b o t h hormone groups (fig. 4): the maximum inhibitory response was 100% in both groups, and IDs0 values (Estrus: 1.3 X 10 -s M; HCG + Progesterone: 1.2 × 10 -s M) were not significantly different.
4. Discussion
a-Adrenoceptor sensitivity did not vary between the hormone groups in this study. This finding is not in accord with the conclusion of Hunter and Kendle (1974) nor of Heilman et al. (1976) who attributed the
effect of progesterone to a decrease in ~adrenoceptor sensivity. However, in these earlier studies a-activity was not monitored exclusively since adrenaline, a mixed agonist with a- and fl-activity, was employed, in the absence of a/3-adrenoceptor blocking agent. In this study, we examined/3-adrenoceptor activity in in vitro preparations in a system of functional antagonism by determining the/3inhibitory effect of (+)-isoprenaline on the excitatory response to a cholinergic agonist. In progesterone-treated tissues, isoprenaline inhibited the contractile response to acetylcholine by a b o u t 90% while in Estrous tissues, isoprenaline only inhibited the acetylcholine response by about 30%. While this might be interpreted to indicate increased /3-receptor activity in the GCG + Progesterone group, it was also important to eliminate the possibility that there might be some change in the intrinsic capacity of the isthmic smooth muscle to relax. However, papaverine, a nonspecific smooth muscle relaxant, exhibited identical dose--response curves in both hormone groups, and thereby demonstrated that the capacities of tissues from both hormone groups to relax were similar. The IDs0 values for the isoprenaline inhibition of the acetylcholine response determined in this study were in close agreement with those quoted b y Hodgson and Pauerstein (1975) for rabbit isthmus. Thus, while the maximum inhibitory effect of isoprenaline was greater in the HCG + Progesterone group than in the Estrous group, the potency of isoprenaline on /3-receptors, calculated from normalized curves, did not vary significantly between groups. This indicated that the difference in tissue responses to isoprenaline isoprenaline might reflect a change in number rather than t y p e of receptor. The present study does not answer the question of whether this might be accomplished by an unmasking of, or de novo formation of /3adrenoceptors. However, since progesterone must be administered for at least 24 hours prior to ovulation for its effect on oviduct motility and ovum transport to be realized
PROGESTERONE AND ADRENOCEPTORS IN OVIDUCT
(De Vargas and Pauerstein, 1976), this interval may be required for the synthesis of new receptors. In the comparison of agonists with high and essentially equal efficacies the relative potencies should be the same for all responses mediated by the same receptor type, regardless of the test preparation, provided that experimental conditions are satisfactory (Furchgott, 1972). This has been illustrated by Furchgott for the potencies of adrenaline, noradrenaline and phenylephrine on aadrenoceptors in various tissues and species. In this study, we characterized the ~-adrenoceptors in rabbit oviductal isthmus by listing the order of potency of adrenergic agonists in the presence of blockade of catecholamine uptake processes and of /3-adrenoceptors. The relative potencies of adrenaline, noradrenaline and phenylephrine were similar to those quoted by Furchgott (1972) for aadrenoceptors in other rabbit tissues and in other species. Thus, the a-adrenoceptor in rabbit oviductal isthmus is apparently identical or very similar to that in other tissues in the rabbit. The order of potency for adrenergic agonists on fl-receptors of rabbit isthmus was similar to that described by Furchgott (1972) and others (e.g., Coleman and Somerville, 1977) for ~l-receptors. Salbutamol, a selective ~2-agonist (Cullum et al., 1969), was much less potent than adrenaline. Not only was the potency of salbutamol reduced, but so too was its maximum inhibitory effect. Salbutamol is also a partial agonist at cardiac fl~adrenoceptors (Brittain et al., 1970). These findings suggest that the ~-adrenoceptor subtype may be ~1 in nature. The pA2 values for propranolol (Furchgott, 1972) do not adequately distinguish / ~ - a n d ~2-adrenoceptors in rabbit tissues ( ~ 8.8; ~: 6.9, 9.0). The pA2 value (approximately 8.3) determined for propranolol in this tissue in preliminary studies (Rheaume and Paton, 1977b) was of the same order as these published results. However, reported pA2 values for practolol, a specific ~-antagonist,
409
do illustrate differences between ~-adrenoceptor subtypes (~1 6.9; ~2 3.9, < 3.5), in that such low pA: values for the ~2-receptor subtype indicates a largely non-specific antagonism (Furchgott, 1972). The pA= value for practolol {approximately 5.3) determined in preliminary studies (Rheaume and Paton, 1977b) was comparable to the values for the ~l-receptor type. Thus, the calculated pA2 values for propranolol and practolol antagonism of the isoprenaline inhibitory effect are compatible with the conclusion that the ~-adrenoceptor subype in the circular muscle of rabbit isthmus (HCG + Progresterone) is largely ~1 in nature. Tubal contractility is not apparently the absolute regulator of ovum transport (Paton et al., 1978). However, altered ~-adrenoceptor activity is compatible with existing concepts of modification of ovum transport by progesterone since reduced responsiveness to noradrenaline might decrease the isthmic barrier to transport and result in acceleration of ovum movement through the oviduct.
Acknowledgements This work was supported by grants from the Medical Research Council of Canada and World Health Organization (Dr. D.M. Paton) and a Medical Research Council Studentship (D.E. Rheaume).
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410 Furchgott, R.F., 1972, The classification of adrenoceptors, an evaluation from the standpoint of receptor theory, in: Catecholamines, eds. H. Blaschko and E. Muscholl (Springer-Verlag, Berlin) p. 283. Heilman, R.D., R.R. Reo and D.W. Hahn, 1976, Changes in the sensitivity of adrenergic receptors in the oviduct during early gestation, Fertil. Steril. 27,426. Higgs, G.W. and A.H. Moawad, 1974, The effect of ovarian hormones on the contractility of the rabbit oviductal isthmus, Can. J. Physiol. Pharmacol. 52, 74. Hodgson, B.J. and C.J. Pauerstein, 1974, The effect of ovulation on the response of rabbit oviduct to adrenergic agonists in vitro, Biol. Reprod. 10,346. Hodgson, B.J. and C.J. Pauerstein, 1975, Effects of hormonal treatments which alter ovum transport on ~-adrenoceptors of the rabbit oviduct, Fertil. Steril. 26,573. Hunter, D.S. and K.E. Kendle, 1974, The influence of hormonal state on the response of the isolated rabbit oviduct to catecholamines, J. Reprod. Fertil. 4 1 , 2 4 5 . Levy, B. and H.R. Lindner, 1972, The effect of adrenergic drugs on the rabbit oviduct, European J. Pharmacol. 18, 15. Longley, W.J., D.L. Black and C.M. Currie, 1968, Oviduct circular muscle response to drugs related
D.E. RHEAUME, D.M. PATON to the autonomic nervous system, J. Reprod. Fertil. 17, 95. Luna, L.G. (ed.), 1968, Manual of Histological Staining Methods (McGraw-Hill, New York). Martin, J.E., R.W. Ware, R.J. Crosby and C.J. Pauerstein, 1970, Demonstration of beta adrenergic receptors in the rabbit oviduct, Gynecol. Invest. 1, 82. Paton, D.M. and A. Johns, 1975, Effects of prostaglandins E 2 and indomethacin on responses of rabbit oviduct to norepinephrine and transmural stimulation, Res. Commun. Chem. Pathol. Pharmacol. 11, 15. Paton, D.M., J.H. Widdicombe, D.E. Rheaume and A. Johns, 1978, The role of the adrenergic innervation of the oviduct in the regulation of mammlian ovum transport, Pharmacol. Rev. (in press). Pauerstein, C.J., V. Anderson, M.L. Chatkoff and B.J. Hodgson, 1974, Effect of estrogen and progesterone on the time-course of tubal ovum transport in rabbits, Amer. J. Obstet, Gunecol. 120,299. Rheaume, D.E. and D.M. Paton, 1977a, Effect of hormonal pretreatment on adrenoceptor sensitivity in the isthmus of rabbit oviduct, Proc. West. Pharmacol. Soc. 20, 19. Rheaume, D.E. and D.M. Paton, 1977b, Studies on ~-receptor activity in the isthmic circular muscle of rabbit oviduct, Can. Fed. Biol. Sci. 20, 122.
403
E F F E C T OF P R O G E S T E R O N E ON A D R E N O C E P T O R S IN THE I S T H M U S OF THE R A B B I T OVIDUCT DORIANNE E. RHEAUME and DAVID M. PATON * Department of Pharmacology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada
Received 20 December 1977, accepted 2 January 1978
D.E. RHEAUME and D.M. PATON, Effect of progesterone on adrenoceptors in the isthmus of the rabbit oviduct, European J. Pharmacol. 48 (1978) 403--410. The effect of adrenergic agonists on the contractility of circular muscle of the isthmus of the oviduct was studied in isolated tissues from rabbits in estrus and after progesterone pretreatment, a-Adrenoceptor sensitivity was not altered by progesterone, and the order of potencies of a-agonists was similar to that for a-adrenoceptors in other rabbit tissues. Progesterone pretreatment increased the maximal inhibitory effect of isoprenaline on ~-adrenoceptors, but did not alter sensitivity to the agonist, suggesting that there had been an increase in the number of ~-adrenoceptors rather than a change in their affinity. The order of potencies of ~-agonists was similar to that for the ~l-adrenoceptor in other rabbit tissues.
Rabbit oviduct
Adrenoceptors
Estrus
1. I n t r o d u c t i o n T h e p r e s e n c e o f a - e x c i t a t o r y a n d ~-inhibit o r y a d r e n o c e p t r o s in r a b b i t o v i d u c t is well e s t a b l i s h e d ( L o n g l e y et al., 1 9 6 8 ; Martin et al., 1970; Levy and Lindner, 1972; Hodgson and Pauerstein, 1974, 1975). a-Adrenoceptor a c t i v i t y p r e d o m i n a t e s u n d e r all h o r m o n a l cond i t i o n s since a d r e n a l i n e a n d n o r a d r e n a l i n e a l w a y s elicit c o n t r a c t i l e r e s p o n s e s t h a t are blocked by phentolamine or phenoxybenzamine. N o r a d r e n a l i n e h a d a d e c r e a s e d p o t e n c y on t h e circular m u s c l e o f t h e i s t h m u s o f r a b b i t o v i d u c t in p r o g e s t e r o n e - d o m i n a n t as c o m p a r e d to e s t r o u s tissues (Higgs a n d M o a w a d , 1974; Rheaume and Paton, 1977a). Inhibition o f c a t e c h o l a m i n e u p t a k e processes did n o t * Present address and to whom correspondence should be addressed: Department of Pharmacology and Clinical Pharmacology, School of Medicine, University of Auckland, Auckland, New Zealand.
Isoprenaline
Progesterone
Noradrenaline
e l i m i n a t e t h e s e p o t e n c y d i f f e r e n c e s since, in t h e p r e s e n c e o f cocaine, h y d r o c o r t i s o n e or b o t h agents, n o r a d r e n a l i n e was sill less p o t e n t in p r o g e s t e r o n e - d o m i n a n t tissues ( R h e a u m e a n d P a t o n , 1 9 7 7 a ) . A r e d u c t i o n in a - a d r e n o c e p t o r s a c t i v i t y ( H u n t e r a n d Kendle, 1 9 7 4 ; H e i l m a n et al., 1 9 7 6 ) or, c o n v e r s e l y , an increase in ~ - a d r e n o c e p t o r a c t i v i t y (Martin et al., 1 9 7 0 ; H o d g s o n a n d Pauerstein, 1 9 7 4 , 1 9 7 5 ) have b e e n p r o p o s e d to e x p l a i n e f f e c t s o f p r o g e s t e r o n e o n r a b b i t o v i d u c t a l isthmus. Such changes in t h e relative sensitivity o f t h e t w o a d r e n o c e p t o r s w i t h changes in n o r m a l d o m i n a n c e ma3 b e o f p h y s i o l o g i c a l signific a n c e since a d m i n i s t r a t i o n o f p r o g e s t e r o n e before ovulation accelerated ovum transport in t h e r a b b i t ( P a u e r s t e i n et al., 1 9 7 4 ) . In t h e p r e s e n t s t u d y we have investigated t h e e f f e c t o f adrenergic agonists o n t h e cont r a c t i l i t y c,,f isthmic circular m u s c l e o f r a b b i t s in estrus a n d a f t e r a p r o g e s t e r o n e - p r e t r e a t m e n t schedule k n o w n to a c c e l e r a t e o v u m t r a n s p o r t ( P a u e r s t e i n et al., 1 9 7 4 ) a n d have studied t h e
404 effects of these hormonal states on a- and fladrenoceptor activity, a- and fi-adrenoceptors in oviductal isthmus were also characterized by determining the order of potency of adrenergic agonists on each receptor system. A preliminary account of this research has been published (Rheaume and Paton, 1977b).
2. Materials and methods
2.1. Animals and hormone pretreatment Mature, female New Zealand white rabbits (3--5 kg) were emplyed. Two hormone groups were studied: Group I, Estrus received 17/3estradiol (5 pg, s.c. daily) for 6 days; Group II, Human Chrionic Gonadotropin (HCG)+ Progesterone Pretreatment received progesterone (2.5 mg, i.m.) on the day of HCG injection (100 iu, i.v.) and on each of the two preceding days. This treatment accelerated ovum transport in the rabbit (Pauerstein et al., 1974). Rabbits in Group II were killed 20 h after HCG administration.
2.2. Measurement o f contractile responses Rabbits were killed by cervical dislocation. After the oviducts had been dissected free of surrounding tissue, the isthmus was identified and cut into 2--3 mm pieces. Oviductal segments were threaded twice through the lumen, and attached at the b o t t o m of the tissue bath and to a transducer. These circular muscle preparations were set up under 0.5 g resting tension in baths containing modified Krebs solution at 28°C, pH 7.4 and bubbled with 95% 02--5% CO2. Tissues were allowed to equilibrate for at least 45 min before any experimental procedure was begun. The modified Krebs solution had the following composition (mM): NaC1 116, KC1 5; CaC12 1.25; MgC12 1.2; NaH2PO4 1.2; NaHCO3 22; D-glucose 11.2. Ascorbic acid, 0.1 mM and Na2EDTA, 0 . 0 4 m M were added to prevent oxidation of amines. Responses to adrenergic agonists were
D.E. RHEAUME, D.M. PATON measured as changes in isometric tension with Grass (FT.03C) Force Displacement Transducers and displayed on a Grass (Model 5D) Polygraph. Responses were allowed to reach a maximum before the drugs were washed out.
2.3. Determination o f hormonal status Uterine histology was examined to define hormonal status, using Johnson's Method for Metachromasia (0.1% toluidine blue) (Luna, 1968). In the Estrous group the endometrium exhibited a proliferative phase; HCG + Progesterone-pretreated endometrium exhibited a secretory phase.
2.4. Dose--response determinations and EDso calculations Non-cumulative concentration-response curves to (--)-adrenaline, (--)-noradrenaline and (--)-phenylephrine were determined using at least 5 animals for each study. Responses were expressed as a percentage of the maximal responses of the tissue (to 10 -4 M adrenaline). Potencies of adrenergic agonists were compared in terms of EDs0 values, which were extrapolated by computer regression analysis of values between 20--80% on the log concentration/response curves. Relative potencies of agonists were expressed as the ratio of EDs0 values, taking the potency of (--)-noradrenaline as 1.0. The effects of various agents on t h e potencies of the adrenergic agonists were examined in dose--response determinations performed after a 20 min exposure to the drug. All drugs were present during the dose--response determination and comparison was made with a control tissue in each experiment.
2.5. a-Adrenoceptor activity The order of potency of adrenergic agonists on ~-adrenoceptors was also determined. Catecholamine uptake processes were blocked with cocaine (3 X 10 -s M) and hydrocortisone (10 -4 M), and t3-adrenoceptors were blocked with propranolol (3.8 X 10 -6 M).
PROGESTERONE AND ADRENOCEPTORS IN OVIDUCT
405
2.6. Characterization of [J-adrenoceptors
2.7. Drugs and chemicals
2.6.1. {J-Adrenoceptor activity in estrous and HCG + progesterone pretreated isthmus fl-Adrenoceptor activity was determined by examining the (+)-isoprenaline antagonism of acetylcholine contractile responses using a modification of the method described b y Hodgson and Pauerstein (1975). At concentrations greater than 10-SM, (+)-isoprenaline exhibits a-excitatory activity in this tissue. Tissue were therefore exposed to phenoxybenzamine (10 -6 M) for 20 min to block aadrenoceptors. Cocaine (3 X 10 -s M) and hydrocortisone (10-4M) were included to block neuronal and extraneuronal uptake of catecholamines. Contractile responses to a maximal dose of acetylcholine (5 X 10 -4 M) were elicited every 15 min. Then, 2 min before the addition of acetylcholine, (+)-isoprenaline (2 X 10 -7 to 10 -4 M) was administered. The % inhibition of the acetylcholine response was calculated b y comparing the magnitude of the response in the presence of (+)-isoprenaline to the mean of control responses before and after. The maximum inhibition of response as well as IDs0 values were compared between hormone treatment groups.
The following drugs were used in this study: (+)-salbutamol sulphate * (Allen & Hanburys, Ltd.}; p r a c t o l o l * and propranolol hydrochloride * (Ayerst, McKenna & Harrison, Ltd.); cocaine hydrochlride (British Drug House, Ltd.), 1713-estradiol (Calbiochem); papaverine hydrochloride (Eli Lilly & Co.); acetylcholine hydrochloride, (--)-adrenaline bitartrate, human chorionic gonadotropin, hydrocortisone 21-sodium succinate, (+)isoprenaline hydrochloride, (--)-phenylephrine hydrochloride, progesterone (Sigma Chemical Co.), phenoxybenzamine hydrochloride * (Smith, Kline & French, Ltd.). Compounds indicated with an asterisk were generously donated by the companies listed.
2.6.2. Order of agonist potency on [J. adrenoceptors A series of experiments as described above was completed for the antagonism by (--)adrenaline, (--)-noradrenaline and (+)-salbutamol of the acetylcholine response in Estrous and HCG + Progesterone-pretreated tissues. The order of agonist potency on fl-adrenoceptors was ascertained and the maximum inhibitory effects of the agonists noted. 2.6.3. Non-specific inhibition of the acetylcholine response Dose--response curves for papaverine inhibi. tion of the cholinergic contractile response were similarly studied in the two hormone groups. The IDs0 values and maximum inhibitory responses were compared between groups.
2.8. Statistical analysis Resulting were expressed as the mean + S.E.M. Student's unpaired two-tailed t-test was employed to make comparative assessments. 95% was chosen as an acceptable level of significance.
3. Results
Responses of tissues were studied at 28°C and in the presence of a reduced concentration of Ca 2÷ (1.25 mM) because spontaneous activity was considerably reduced under these conditions, and responses to agonists and to transmural stimulation did not display desensitization with time. At 37°C there was a timedependent reduction in responses to both (--)-noradrenaline and to transmural stimulation, possibly due to prostaglandin liberation as the reduced responsiveness was abolished by indomethacin (Paton and Johns, 1975). 1
3.1. a-Adrenoceptor sensitivity a-Adrenoceptor sensitivity did not vary between groups since the potency of (--)phenylephrine in the presence of propranolol
406
D.E. R H E A U M E , D.M. P A T O N A
B
3.2. Characteristics of [J-adrenoceptors
10
10
Fig. 1. a - A d r e n o c e p t o r sensitivity of rabbit oviductal isthmus in Estrous (open bar) and HCG + Progesterone-pretreated (hatched bar) tissues. The histogram represents the EDs0 for: (A) (--)-phenylephrine (in the presence of 3.8 x 10 .6 M propranolol); and (B) for (--)-adrenaline (in the presence of 3 × 10 -s M cocaine, 10 -4 M h y d r o c o r t i s o n e and 3.8 × 10 .6 M propranolol). Mean ± S.E.M. of 5--6 observations. Ordinate: (A) EDs0 (×10 -6 M); (B) EDs0 (x 10 -7 M).
(3.8 X10-6 M) and the potency of (--)adrenaline in the presence of c o c a i n e + hydrocortisone + propranolol were not altered by either of the hormonal treatments (fig. 1). The order of p o t e n c y for the adrenergid agonists on a-adrenoceptors (i.e., in the presence of cocaine + hydrocortisone + propranolol) was determined to be (--)-adrenaline > (--)-noradrenaline > (--)-phenylephrine, the EDs0 values being 5.0 X 10 -7 M, 6.8 X 10 -7 M and 3.8 X 10 .6 M respectively. In table 1 the relative potencies for the adrenergic agonists on a-adrenoceptors in oviductal isthmus are compared with values for other rabbit tissues.
3.2.1. ~-Adrenoceptor activity in Estrous and HCG ÷ Progesterone-pretreated isthmus (+)-Isoprenaline antagonism of the acetylcholine-elicited contractile response was studied to provide a measure of fi-adrenoceptor activity in Estrous and HCG + Progesteronepretreated isthmus. In progesterone-pretreated tissues, the ~-agonist maximally inhibited the contractile response to acetylcholine by about 90%, while in Estrous tissues, (+)-isoprenaline only maximally inhibited the acetylcholine response by about 30% (fig. 2). Propranolol (3.8 X 1 0 -6 M) blocked the effect of (+-)-isoprenaline in both groups. The absolute magnitude of the contractile response to acetylcholine (5 X 10 -4 M) was significantly greater in Estrous tissues (0.42 + 0.05 g, n = 7) than in HCG + Progesteronepretreated tissues (0.27 + 0.03 g, n = 13). In spite of this, the absolute magnitude of the inhibition of the acetylcholine response produced by (+)-isoprenaline was still greater in the HCG + Progresterone group (0.24 g) than in the Estrous group (0.13 g). Normalized curves for the inhibitory effect of (+)-isoprenaline were also determined by expressing each inhibitory response as a % of the maximum inhibitory response for each treatment group, and b y plotting against (+-)isoprenaline concentration (fig. 3). When expressed in this manner, the IDs0 values were not found to differ between the Estrous
TABLE 1 Relative potencies of adrenergic agonists on a-adrenoceptors in various rabbit tissues. Tissue
r. Thoracm aorta 1 Duodenum 1 Stomach fundus 1 Oviductal isthmus (estrus)
1 F r o m F u r c h g o t t , 1972.
Relative potencies (--)-Adrenaline
(--)-Phenylephrine
(--)-Noradrenaline
1.2 2.0 1.0 1.4
0.2 0.25 0.25 0.2
1 1 1 1
P R O G E S T E R O N E A N D A D R E N O C E P T O R S IN O V I D U C T
407
I00
I00
80
80
60
60 40
40
20
2O
0 0
I
I
I
10-6
I0-~
10-4
I
2 xlO-7
L 2xl()-7
~-6
~[s
~-4
Fig. 2. ~ - A d r e n o c e p t o r activity o f r a b b i t o v i d u c t a l i s t h m u s in E s t r o u s a n d HCG + P r o g e s t e r o n e - p r e t r e a t e d i s t h m u s . Each p o i n t r e p r e s e n t s t h e m e a n ± S.E.M. o f 6--8 o b s e r v a t i o n s o f t h e % i n h i b i t i o n o f t h e a c e t y l c h o l i n e c o n t r a c t i l e r e s p o n s e b y (±)-isoprenaline. E s t r o u s group, o o; HCG + Progesterone, • =. O r d i n a t e : % i n h i b i t i o n o f r e s p o n s e t o a c e t y l c h o l i n e (5 x 10 -4 M). Abscissa: i s o p r e n a l i n e c o n c e n t r a t i o n (M).
Fig. 3. N o r m a l i z e d curves for t h e i n h i b i t o r y e f f e c t o f (+-)-isoprenaline o n t h e a c e t y l c h o l i n e r e s p o n s e in Est r o u s a n d H C G + P r o g e s t e r o n e - p r e t r e a t e d tissues. Each i n h i b i t o r y r e s p o n s e is e x p r e s s e d as a % o f t h e m a x i m u m i n h i b i t o r y r e s p o n s e for t h a t h o r m o n e g r o u p -+S.E. E s t r o u s group, o o; H C G + P r o gesterone, • =. 6--8 o b s e r v a t i o n s . O r d i n a t e : % m a x i m u m i n h i b i t o r y r e s p o n s e ; abscissa: i s o p r e n a l i n e c o n c e n t r a t i o n (M).
and HCG + Progesterone groups being 3.4 X 10 -7 M and 5.2 X 10 -7 M respectively. Thus, while the maximum inhibitory effect was greater in the HCG + Progesterone group, the potency of (+)-isoprenaline on ~-adrenoceptors did not vary.
ceptors in HCG + Progesterone-pretreated tissues was (+ )-isoprenaline /> (--)-noradrenaline > (--)-adrenaline ~ (+)-salbutamol, the EDs0 values being 5.1 X 10 -TM, 5.2 × 10 -TM, 1.3 X 1 0 -6 M a n d > 1 0 -4 M respectively. Table 2 lists the potencies for the adrenergic agonists on fi-adrenoceptors in oviductal isthmus along with comparable values for other rabbit tissues. (--)-Adrenaline and (--}-noradrenaline produced maximum inhibitory
3.2.2. Order o f agonist potency on fiadrenocep tors The order of agonist potency on 13-adreno-
TABLE 2 Relative p o t e n c i e s of a d r e n e r g i c agonists o n ~3-adrenoceptors in various r a b b i t tissues. Tissue
Thoracic aorta 1 Left atrium 1 Small i n t e s t i n e 1 Stomach fundus I Oviductal isthmus (HCG + P) 1 F r o m F u r c h g o t t , 1972.
Relative p o t e n c i e s (--)-Isoprenaline
(--)-Adrenaline
(--)-Noradrenaline
(+)-Salbutamol
130 3.5 1.5 2.5 1.0
65 0.5 0.2 1.2 0.4
1 1 1 1 1
< 0.01
408
D.E. R H E A U M E , D.M. P A T O N
10C
8C
60
4C
i
3 × 1 0 -'s
l
10-5
I
3 x 1 0 -5
I
10 -4
i
3 x 1 0 -3
Fig. 4. I n h i b i t o r y e f f e c t of p a p a v e r i n e o n t h e acetylc h o l i n e response in E s t r o u s a n d H C G + P r o g e s t e r o n e p r e t r e a t e d r a b b i t isthmus. Each p o i n t r e p r e s e n t s the m e a n % i n h i b i t i o n _+S.E. o f 5 observations. Estrus, o o; H C G + P r o g e s t e r o n e , • ~. O r d i n a t e : % i n h i b i t i o n o f r e s p o n s e t o a c e t y l c h o l i n e (M); abscissa: p a p a v e r i n e c o n c e n t r a t i o n (M).
effects of the same order of magnitude as (+)isoprenaline. However, (+)-salbutamol, a specific ~2-agonist, failed to achieve such a maximum effect, and at 1 0 - 4 M elicited only 40--50% of the maximum inhibitory response to (+)-isoprenaline.
3.2.3. Non-specific inhibition o f the acetylcholine response Papaverine exhibited identical dose-response curves in b o t h hormone groups (fig. 4): the maximum inhibitory response was 100% in both groups, and IDs0 values (Estrus: 1.3 X 10 -s M; HCG + Progesterone: 1.2 × 10 -s M) were not significantly different.
4. Discussion
a-Adrenoceptor sensitivity did not vary between the hormone groups in this study. This finding is not in accord with the conclusion of Hunter and Kendle (1974) nor of Heilman et al. (1976) who attributed the
effect of progesterone to a decrease in ~adrenoceptor sensivity. However, in these earlier studies a-activity was not monitored exclusively since adrenaline, a mixed agonist with a- and fl-activity, was employed, in the absence of a/3-adrenoceptor blocking agent. In this study, we examined/3-adrenoceptor activity in in vitro preparations in a system of functional antagonism by determining the/3inhibitory effect of (+)-isoprenaline on the excitatory response to a cholinergic agonist. In progesterone-treated tissues, isoprenaline inhibited the contractile response to acetylcholine by a b o u t 90% while in Estrous tissues, isoprenaline only inhibited the acetylcholine response by about 30%. While this might be interpreted to indicate increased /3-receptor activity in the GCG + Progesterone group, it was also important to eliminate the possibility that there might be some change in the intrinsic capacity of the isthmic smooth muscle to relax. However, papaverine, a nonspecific smooth muscle relaxant, exhibited identical dose--response curves in both hormone groups, and thereby demonstrated that the capacities of tissues from both hormone groups to relax were similar. The IDs0 values for the isoprenaline inhibition of the acetylcholine response determined in this study were in close agreement with those quoted b y Hodgson and Pauerstein (1975) for rabbit isthmus. Thus, while the maximum inhibitory effect of isoprenaline was greater in the HCG + Progesterone group than in the Estrous group, the potency of isoprenaline on /3-receptors, calculated from normalized curves, did not vary significantly between groups. This indicated that the difference in tissue responses to isoprenaline isoprenaline might reflect a change in number rather than t y p e of receptor. The present study does not answer the question of whether this might be accomplished by an unmasking of, or de novo formation of /3adrenoceptors. However, since progesterone must be administered for at least 24 hours prior to ovulation for its effect on oviduct motility and ovum transport to be realized
PROGESTERONE AND ADRENOCEPTORS IN OVIDUCT
(De Vargas and Pauerstein, 1976), this interval may be required for the synthesis of new receptors. In the comparison of agonists with high and essentially equal efficacies the relative potencies should be the same for all responses mediated by the same receptor type, regardless of the test preparation, provided that experimental conditions are satisfactory (Furchgott, 1972). This has been illustrated by Furchgott for the potencies of adrenaline, noradrenaline and phenylephrine on aadrenoceptors in various tissues and species. In this study, we characterized the ~-adrenoceptors in rabbit oviductal isthmus by listing the order of potency of adrenergic agonists in the presence of blockade of catecholamine uptake processes and of /3-adrenoceptors. The relative potencies of adrenaline, noradrenaline and phenylephrine were similar to those quoted by Furchgott (1972) for aadrenoceptors in other rabbit tissues and in other species. Thus, the a-adrenoceptor in rabbit oviductal isthmus is apparently identical or very similar to that in other tissues in the rabbit. The order of potency for adrenergic agonists on fl-receptors of rabbit isthmus was similar to that described by Furchgott (1972) and others (e.g., Coleman and Somerville, 1977) for ~l-receptors. Salbutamol, a selective ~2-agonist (Cullum et al., 1969), was much less potent than adrenaline. Not only was the potency of salbutamol reduced, but so too was its maximum inhibitory effect. Salbutamol is also a partial agonist at cardiac fl~adrenoceptors (Brittain et al., 1970). These findings suggest that the ~-adrenoceptor subtype may be ~1 in nature. The pA2 values for propranolol (Furchgott, 1972) do not adequately distinguish / ~ - a n d ~2-adrenoceptors in rabbit tissues ( ~ 8.8; ~: 6.9, 9.0). The pA2 value (approximately 8.3) determined for propranolol in this tissue in preliminary studies (Rheaume and Paton, 1977b) was of the same order as these published results. However, reported pA2 values for practolol, a specific ~-antagonist,
409
do illustrate differences between ~-adrenoceptor subtypes (~1 6.9; ~2 3.9, < 3.5), in that such low pA: values for the ~2-receptor subtype indicates a largely non-specific antagonism (Furchgott, 1972). The pA= value for practolol {approximately 5.3) determined in preliminary studies (Rheaume and Paton, 1977b) was comparable to the values for the ~l-receptor type. Thus, the calculated pA2 values for propranolol and practolol antagonism of the isoprenaline inhibitory effect are compatible with the conclusion that the ~-adrenoceptor subype in the circular muscle of rabbit isthmus (HCG + Progresterone) is largely ~1 in nature. Tubal contractility is not apparently the absolute regulator of ovum transport (Paton et al., 1978). However, altered ~-adrenoceptor activity is compatible with existing concepts of modification of ovum transport by progesterone since reduced responsiveness to noradrenaline might decrease the isthmic barrier to transport and result in acceleration of ovum movement through the oviduct.
Acknowledgements This work was supported by grants from the Medical Research Council of Canada and World Health Organization (Dr. D.M. Paton) and a Medical Research Council Studentship (D.E. Rheaume).
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