Prompt Effect of Progesterone on the Adrenergic Response of Smooth Muscles Shigeru MORISHITA Departmentof Pharmacology,Kawasaki Medical School, Kurashiki,Okayama 701 -01, Japan AcceptedJuly 7, 1986 Abstract-1) The contractile effects of epinephrine on the uterus and ductus deferens of the rabbit and the ductus deferens of the monkey were inhibited by the preincubation with progesterone (6.4X10-5 M) for 1 or 3 min in Locke-Ringer solution. Epinephrine relaxed the guinea pig uterus and taenia caecum. The relaxant effects were enhanced by preincubation with progesterone. Their effects were in a dose-dependent manner. 2) There was no apparent change in the number and affinity of alpha-adrenergic receptors in the uterus of rabbits and the ductus deferens of guinea pigs during the incubation with progesterone. Proges terone has no direct effect on alpha-adrenergic receptors. 3) All smooth muscles yielded reproducible contractile reactions to Ca2+ when maintained in depolarizing Tyrode's solution containing K+ (40 mmol/I). Their concentration-response curves were inhibited by preincubation with progesterone (6.4X10-5 M), and they were shifted to the right in a concentration-dependent manner. Established Ca2+ induced contractions were rapidly relaxed by the addition of progesterone (6.4x10-5 M). 4) It suggests that progesterone directly affects the plasma membrane and inhibits the voltage-dependent Ca 21 channel and then inhibits smooth muscle contraction. The hypothesis of a universal mechanism and monkey in relation to the adrenergic of steroid hormone action has been proposed mechanism and the Ca2+ channel. by Thompson and Lippman (1). Although steroids have a classical receptor-mediated Materials and Methods pathway of hormonal action, it is not sufficient Materials: The following drugs were used: to account for all the known effects of steroids. 4-Pregnene-3,20-dione (progesterone), Sig For example, Baulieu and co-workers (2) ma Chemical Co.; epinephrine, Daiichi have demonstrated that progesterone and Chemical Co.; phentolamine mesylate (phen other steroid molecules can promote the tolamine), Ciba Gaigy; dl-propranolol hydro maturation of Xenopus laevis oocytes, chloride (propranolol), Wako Pure Chemical although these cells do not contain steroid Ind.; 3H-dihydroergocryptine and ACSII receptors. Kaya and Saito (3) have recently scintillation mixture, Amersham; All other demonstrated that steroids have a direct, chemicals were purchased from Wako Pure non-genomic effect on the erythrocyte mem Chemical Ind. Progesterone (1 mg) dissolved brane. Therefore, it seems that, in addition to in propylene glycol (0.1 ml) was used in the the receptor-mediated mechanism, steroids Magnus method. Progesterone dissolved in may also operate through other mechanisms 2% ethanol was used in the receptor assay. and, in particular, through an effect on the Deionized distilled water was used in making plasma membrane. In the present paper, I up other solutions. Propylene glycol up to examined the direct, non-genomic effects of 0.1-0.3 ml had no effect on the excitability progesterone on the plasma membrane with of smooth muscles. The 2% ethanol used as a the smooth muscle of the guinea pig, rabbit vehicle had no effect in the receptor assay. Methods: The uterus, ductus deferens and used immediately. taenia caecum (2 3 cm) were dissected from Receptor assay: The assay was done Hartley guinea pig, New Zealand White according to the method of Williams and co rabbit, Flemish giant rabbit and Japanese workers (4). stumptailed macaque Macaca fuscatx. Radioligand: 3H-Dihydroergocryptine was Organs were cleaned of surrounding chosen as the a-adrenergic radioligand for connective tissue, vessels and fat tissue and this study. The reason for using 3H-di set up in a 50 ml Magnus organ bath con hydroergocryptine to identify a-adrenergic taining modified Locke-Ringer solution or receptors was the high potency of dihydro K+-depolarizing Tyrode's solution maintained ergocryptine as an a-adrenergic antagonist. at 37'C and gassed with air. The com Its apparent affinity for a-adrenergic receptors position of the modified Locke-Ringer as determined from pharmacological studies solution (Locke-Ringer solution) was NaCI, is greater than that of any other ergot 154; KCI, 15.6; CaC12, 2.2; M9 C12, 0.98; alkaloid (4). NaHCO3, 11.9; NaH2PO4, 0.4; glucose, 5.5 Membrane preparation: Uterus was ob mM; pH 7.1. K+-depolarizing Tyrode's tained from rabbit, and ductus deferens was solution (K+-Tyrode's solution) was NaCI, obtained from guinea pig. The tissues were 97; KCI, 40; NaHCO3, 11.9; NaH2PO4, 0.4; removed and cleaned of surrounding connec glucose, 5.5 mM; pH 7.1. Contractile and tive tissues in ice cold buffer (0.25 M relaxant responses were measured under sucrose, 1 MM MgC12, 5 mM Tris-HCI, pH isotonic conditions (1 g) using a Nihon 7.4). The tissues were homogenized in 4-5 Kohden isotonic transducer connected to an volumes of the same buffer using a Polytron EGB36069 recorder. When maintained in PT-10 homogenizer at setting 6 for four 10-s Locke-Ringer solution, cumulative concen periods at 0°C. After filtration through a tration-response curves were obtained to single layer of gauze, the homogenate was epinephrine (0.1-100 ,ug/50 ml) by in centrifuged at 400xg for 10 min at 4'C, and creasing the concentration in logarithmic the pellet was discarded. The supernatant increments. To observe the effect of pro was centrifuged at 28000xg for 10 min at gesterone on the Ca2+ channel, K+-Tyrode's 4°C. The resulting pellet was suspended in solution was used. When maintained in K+ the incubation buffer (10 mM MgC12, 50 mM Tyrode's solution, cumulative concentration Tris-HCI, pH 7.5) for use in the binding response curves were obtained to CaC12 assay. (0.03-30 mM) by increasing the Ca2+ con Binding assay: 3H-Dihydroergocryptin centration in logarithmic increments. The (3H-DHE), 0.015 ml (specific activity 15.1 100% response was taken as the maximum Ci/mmol) and the membrane preparation, response (contraction or relaxant) at the 0.1 ml (3 mg protein/ml) were incubated higher epinephrine concentration, and each together for 15 min at 25°C with shaking in response was taken at 1 or 3 min after the a total volume 0.15 ml. Incubations were addition of epinephrine to the bath. Pro terminated by diluting the 0.15 ml incubation gesterone 1 mg/50 ml (6.4x10-5 M) was mixtures with 2.5 ml of incubation buffer added to the bath at 1 min before the (25°C) followed by a rapid filtration through application of catecholamine or CaC12, and a TOYO GC50 glass fiber filter. Filters were concentration-response curves of catechol rapidly washed with 20 ml of incubation amine or CaC12 were measured. The time buffer (25°C). After drying, the filters were required for relaxation of the Ca2+-contracted removed to vials. Five ml of ACS II muscle after the addition of progesterone was (Amersham) scintillation mixture were added compared with the control in which the to each vial, and tritium was assayed by relaxant time was measured after the wash measuring the radioactivity of each sample out. The bath fluid was exchanged each time for 10 min in a liquid scintillation spec for fresh solution, and the organs were then trometer (LSC-900). Counting efficiencies returned passively to their resting tension. were 40%. Nonspecific binding is defined as Epinephrine was dissolved each time and binding under 0.01 mM phentolamine. Fig. 1. Scatchard analysis of 3H-DHE binding in rabbit uterus. Lines were determined by linear regression analysis (r=0.82). Specific binding is defined as the total radioactivity bound minus the nonspecific binding. Membrane preparations were Fig. 2. Effects of progesterone on cumulative con assayed with progesterone (1.31 x10-5 M) centration response curves with epinephrine on in 2% ethanol, and then the binding sites and smooth muscles in Locke-Ringer solution. Control affinity (K,) compared with the control. (J) and progesterone (6.4X10-5 M) added before Progesterone of more than 1.31 x10-5 M the application of epinephrine (A). Progesterone could not be dissolved in 2% ethanol. The displaced the curves with epinephrine in rabbit protein content of the final suspension was uterus, rabbit ductus deferens and monkey ductus determined by the method of Lowry et al. (5). deferens to the right. In contrast, the curves with Student's t-test was used for comparison of epinephrine in guinea pig uterus, guinea pig taenia the mean values. The data from each experi caecum and guinea pig ductus deferens were ment were analyzed by a Scatchard plot. shifted to the left. (Fig. 1). Results Progesterone (6.4x10-5 M) added to the Contractile effect of epinephrine in Locke bath 1 or 3 min before the application of Ringer solution: The uterus (rabbit) and the epinephrine inhibited the contract le effect of ductus deferens (rabbit, monkey and guinea epinephrine on the rabbit uterus and the pig) were contracted by epinephrine in ductus deferens of rabbit and monkey. The Locke-Ringer solution. The uterus and taenia dose-response curves were shifted to the caecum from guinea pig were relaxed. right (Fig. 2 a, b, c). However, progesterone Those concentration-response curves were enhanced the relaxant effect of epinephrine reproducible and dose-dependent. (Fig. 2) on the uterus and taenia caecum of guinea These responses did not change under 0.01 pig. In these cases, the dose-response curves mM propranolol, but were greatly reduced by were shifted to the left (Fig. 2 d, e). Proges 0.01 mM phentolamine. terone enhanced the contractile effect of The effects of progesterone on the con epinephrine on the ductus deferens of guinea tractile or relaxant effects of epinephrine: pig.