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EFFECTS OF HIGH POTASSIUM DEPOLARIZATION, CARBACHOL AND OXYTOCIN ON THE LANTHANUM-RESISTANT 45Ca FRACTION IN RAT MYOMETRIUM

The effects of high potassium, carbachol and oxytocin on 40Ca-45Ca exchange in the estradiol-treated rat uterine longitudinal muscle were compared with those in the ovariectomized one using the modified "Lanthanum Method." The treatment of estro genized or ovariectomized myometrium with 60 mM K+ significantly increased the 45Ca space at 5-, 10-, 30 and 60-min incubation times. The increase in the 45Ca by highK+ in the estrogenized myometrium was much greater than that in the ovariectomized one. Carbachol (2•~10-6 M) had little influence on the 45Ca space at all incubation periods with the exception of the 10-min one in both uteri. The higher concentration of carbachol (2•~10-4 M) in the estrogenized myometrium produced a marked increase in the 45Ca space after the 10-min incubation, while in the ovariectomized uterus, a slight increase in the 45Ca space was observed at the 10and 30-min incubation times. The increase in the 45Ca space by 2•~10-4M carbachol in the estrogenized myometrium was greater than that in the ovariectomized one. Oxytocin (10-4 and 10-3 units/ml) increased the 45Ca space at and after the 10-min incubation times in the estrogenized uterus, while in the ovariectomized uterus, oxytocin (10-3 and 10-2 units/ml) showed little influence on the increase in the 45Ca space. The increase in 45Ca space by carbachol and oxytocin was inhibited by papaverine. These results suggest that differences in dependence upon Ca2+ in the medium may exist in contractions induced by high-K+, carbachol and oxytocin in the uterine smooth muscle and that the contractions in the estradiol-treated myometrium may be more dependent upon extracellular Ca2+ than those in the ovariectomized one. It has been reported that the responses of rat uterus to sympathomimetic amines vary with the hormonal state of the individual (1 3), and permeabilities of ions in rat uterine smooth muscle may alter with changes in hormonal influences (4-6). Recently, Uruno et al. (7) presented evidence that the antioxytocin action of papaverine in the estrogenized rat uterus was antagonized by excess external Ca 21 (5-10 mM), but not antagonized in the ovari ectomized one, and Ca exchangeability in the estrogenized myometrium was greater than that in the ovariectomized one. These results suggest that contractile agents may produce different effects on Ca exchange ability in the uterine smooth muscle of rats in different hormonal states. The present study was undertaken to investigate the effects of high potassium, carbachol and oxytocin on 40Ca 45Ca ex change in the estradiol-treated rat uterine longitudinal muscle, and their effects were compared with those in the ovariectomized one using the modified "Lanthanum Method." Materials and Methods Virgin female Wistar rats weighing 150 to 250 g were primed by a subcutaneous in jection of estradiol benzoate (1.0 mg/kg) dissolved in peanut oil two days before sacrifice, or they were ovariectomized by the dorsal route under ether anaesthesia and allowed to recover for more than a week before the experiments. Rats were stunned by a blow on the head, the uterine horns were rapidly removed, and placed in Tris-buffered physiological salt solution. The Tris-buffered solution had the following millimolar composition: NaCI, 125; KCI, 2.7; MgC12, 1.2; CaC12, 1.8; glucose, 11.0; and tris (hydroxymethyl) aminome thane, 23.8. The pH was adjusted to 7.4 with 6N HCI. For measurement of the 45Ca tissue space, the excised uterine horns in Tris-buffered solution, bubbled continuously with 100% 02 at room temperature, were freed from fat and connective tissue; and the myometrial longitudinal muscle layers were separated as described previously (7). After equilibration with the oxygenated Tris-buffered solution for 60 min which was kept at 32'C, the muscle layer was incubated in the oxygen ated Tris-buffered solution (10 ml) containing 45Ca (0 .1 /iCi/ml) or 45Ca plus KCI (60 mM), carbachol (2X 10'6 and 2x10-4 M) or oxytocin (10'4, 10'3 and 10-2 units/ml) for given times (5, 15, 30 and 60 min) at 32°C. In some experiments, an antispasmodic, papaverine (3x10'5 M) or atropine (3x10'6 M), was allowed to remain in contact with the muscle layer 5 min before and throughout the period of the incubation. After the desired time interval, the muscle layer was transferred into a beaker (50 ml) containing the oxygenated La 3+-Tris solution for 5 min. The La3+-Tris solution was made by re placing CaC12 in the Tris-buffered solution by 20 mM LaC13. The purpose of the La 31 treatment for 5 min was to minimize 45Ca loss from smooth muscle cells during the relaxation since the contractile tissue was relaxed by exposure to La3+. The La3 treated tissue was then removed from the La3+ solution, gently blotted on Whatman 42 filter paper, and weighed. After weighing the wet tissue was placed in 1 ml Protosol (New England Nuclear, Massachusetts) and incubated for 3 hr at 55°C to solubilize the tissue. Aliquots of this solution were used for the radioassay of the 45Ca fraction resistant to displacement by 20 mM La3+ (8) by liquid scintilation counting. The 45Ca tissue space was calculated as the ratio between the radioactivity (cpm) of the muscle and that of the bathing medium (9). All experiments were carried out on a pair basis: i.e. one longitudinal muscle layer always served as the control for the other layer. The data were analyzed by means of the Student's t-test, and statistical signifi cance was set at P<0.05. The drugs and chemicals used were: carbachol (Sigma Chemical Co. Ltd., St. Louis), oxytocin (Atonin-0, Teikoku Hor mone MFG. Ltd., Tokyo), papaverine hydro chloride (Sigma Chemical Co. Ltd., St. Louis), atropine sulfate (E. Merck, Darms tadt), lanthanum chloride (Wako Pure Chemical Industries Ltd., Osaka), and 45CaCI2 (s.a.=16.6 mCi/mg Ca, New England Nuclear, Massachussets). All the other chemicals were of reagent grade, and all solutions were made using reagent-grade water processed through Milli-Q systems (Millipore Corp., Bedford).

It has been reported that the responses of rat uterus to sympathomimetic amines vary with the hormonal state of the individual (1 3), and permeabilities of ions in rat uterine smooth muscle may alter with changes in hormonal influences (4)(5)(6).
Recently both myometria ( Fig. 1 A and 1 B). Figure 2 illustrates the effects of carbachol on the 45Ca space in the estrogenized or ovariectomized rat myometrium. The con centrations of carbachol, 2x10-6 M and 2 x 10-4 M, were chosen to give about 50% and maximal contractile responses to the uterine longitudinal smooth muscle strips, respectively. Carbachol at the concentration of 2x10'6 M had little influence on the 45Ca space at all incubation periods except for the 10-min one in both estrogenized and ovariectomized myometrial strips ( Fig. 2A  and 2C). The higher concentration of car bachol (2x`10-4 M) in the estrogenized myometrium produced a marked increase in the 45Ca space at and after the 10-min incubation times, while in the ovariectomized tissue a slight increase in the 45Ca space was observed at the 10 and 30-min incubation times ( Fig. 2B and 2D). The increase in the 45Ca tissue space by 2 x 10-4 M carbachol in the estradiol-treated myometrium was much greater in comparison with that in the ovariectomized one ( Fig. 2B and 2D). : 60 mM Kim. The ordinate represents the 45Ca tissue space (ml/ 100 g wet wt.), and the abscissa represents time in minutes after incubation in the presence or absence of 60 mM Ky. The bar above or below each point is the standard error for 4 to 6 different preparations. The S.E. of the mean is indicated if it is larger than the size of the symbols. `Significantly different from the control at P<0.05. Figure 3 shows the effects of oxytocin on the 45Ca space in the estrogenized or ovari ectomized rat myometrial longitudinal smooth muscle layers. The oxytocin concentrations used were 10-4 and 10-3 units/ml in the estradiol-treated myometrium and 10-3 and 10-2 units/ml in the ovariectomized one because the sensitivity was not the same for the estrogenized and ovariectomized pre parations. The respective concentrations of oxytocin produced approximately 50% and maximal contractile responses in the respec tive tissues.
Oxytocin increased the 45Ca space at and after the 10-min incubation times in the estradiol-primed rat uterine longitudinal smooth muscle. However, the concentration-dependent increase was not observed (Fig. 3A and 3B). In the case of the ovariectomized myometrium, oxytocin at the concentration of 10-3 or 10-2 units/ml showed little influence on the increase in the 45Ca space ( Fig . 3C and 3D).
In order to investigate whether the increase in 45Ca tissue space by carbachol or oxytocin may be related to its contractile response or not the effects of antagonists in the presence or absence of each agonist on Ca exchange ability were studied in the uterine longitudinal smooth muscle layers of estrogen-dominated rats. Figure 4 shows the effects of two different types of antispasmodics, papaverine and atropine, on the 45Ca space in the estradiol-treated myometria. Papaverine     The significant increases in the 45Ca space were observed in both the initial and later phase by isotonic 60 mM K+ in both estradiol-dominated and ovariectomized rat myometria (Fig. 1). The increase in the 45Ca space in the estrogenized myometrium, es pecially in the later phase, was much greater than that in the ovariectomized one. The greater increase in the 45Ca space by high-K+ in the later phase in the estrogenized uterus may be due to the fact that the Ca exchange ability in the estrogenized tissue is larger than that in the ovariectomized one (7). The contractile responses of the rat longitudinal muscle strips to the contractile agents used here reached their maximum within 10 min (data not shown). This implies that the increase in 45Ca space in the initial phase under our experimental conditions may be mainly related to the increase in 45Ca influx, if the contraction is dependent upon extracellular Cat+, while the increase in the later phase is related to the increased Ca exchangeability by the agonists. Therefore, our results suggest that high-K+ may increase Ca2+ influx and Ca exchangeability in the estradiol-treated rat myometrium and that in the ovariectomized uterus, high-K+ may increase Ca 21 influx in the initial phase and induce the smaller increase in Ca exchange ability in the later phase. Ogasawara et al.
(12) reported that the amplitude of the tonic contraction induced by the isotonic K solution in the estradiol-treated rat uterine longi tudinal muscle is larger than that in the ovariectomized one and that the dependence of the K-contracture on the external Ca concentration is most greatly influenced in the estrogen-treated uterus. These facts may be related, in part, to the increased Ca exchangeability in the estradiol-dominated rat uterus.
Atropine had little influence on the increase in 45Ca space in the absence of an agonist, but papaverine significantly inhibited the increase in the 45Ca space (Fig. 4). Further more, papaverine inhibited the increase in the 45Ca space by carbachol or oxytocin, and atropine inhibited the increase in the 45Ca space by carbachol (Fig. 5). Since papa verine did not significantly inhibit the in creased 45Ca space at the 60-min incubation time, it seems likely that papaverine slows Ca exchangeability or Ca influx from the ex tracellular space in the myometrium stimulated by the contractile agents. Our results obtained here suggest that the increase in the 45Ca space by the spasmogens may be related to the contractions induced by them. Papa verine, but not atropine, decreased the 45Ca space in the absence of the agonist (Fig. 4).
This reduced exchangeability of Ca may partly participate in the relaxing or inhibitory action of papaverine in the myometrium. It has been suggested that one of the mecha nisms of action of papaverine may be related to a reduction of Ca influx (13-15) and/or an increase in Ca efflux (13. 15-17) in smooth muscle.
Carbachol at the concentration of 2x10-6 M had little influence upon the 45Ca space in both estrogenized and ovariectomized myometria ( Fig. 2A and 2C). These results suggest that contraction induced by the lower concentration of carbachol may be less dependent upon Ca2+ in the medium in both myometria. The higher concentration of carbachol (2 x 10`4 M) produced a larger increase in the 45Ca space after the 10-min incubation in the estrogenized myometrium than that in the ovariectomized one ( Fig. 2B  and 2D). These results suggest that the later phase of contraction by the higher concen tration of carbachol may be partly dependent upon Ca2+ from an extracellular source. In addition, our results show that the influx of Ca2+ in the initial phase by carbachol is much smaller than that by high-K+.
Oxytocin increased the 45Ca space in both initial (at 10-min incubation time) and later phases in the estrogen-dominated myo metrium, while it had little influence on the increase in the 45Ca space in the ovari ectomized uterus (Fig. 3). These results suggest that the extracellular Ca2+ may participate in contraction by oxytocin to a certain extent in the estrogenized uterus. In the ovariectomized uterus, when the muscle is contracted by oxytocin, the dependence upon Ca2+ in the medium and the increase in Ca exchangeability in the later phase may be very small. Oxytocin did not increase the 45Ca space at the 5-min incubation time in contrast to high-K+ in the estrogenized uterus ( Fig. 3). This indicates that there may be some difference between K+ and oxytocin induced contractions and that the K+-induced contraction may be more dependent upon extracellular Ca2+ than the oxytocin-induced one in the estrogen-dominated uterus. Our results also indicate that contraction induced by oxytocin among the spasmogens used is exceedingly influenced by estrogen treatment in the rat uterine longitudinal smooth muscle. The concentration-dependent increase in the 45Ca space by oxytocin was not observed in the estrogenized uterus. This reason is at present unknown. This, however, raises the possibility that the increased 45Ca fraction by oxytocin might be partly derived from a mobile membrane-bound source which is affected by La3+ In the rat myometrium, several authors (18, 19) reported that it was not possible to demonstrate increased influx of 45Ca as sociated with contraction induced by con tractile agents such as high KCI, acetyl choline or oxytocin. Hodgson and Daniel (19) used pregnant rats and measured 45Ca uptake after a 2 mM-lanthanum wash, while we used the estrogen-dominated and ovariectomized longitudinal myometria of rats and treated the tissue by 20 mM La3+ for 5 min. Thus, there are some differences between their experimental conditions and procedures and ours. This makes it difficult to strictly compare our present results with theirs. The discrepancy between their results and ours might be due to the different hor monal states of animals and/or the possibility that the sites of exchange of 45Ca in the rat myometrium might be located in the cell membrane; therefore, an increased 45Ca fraction(s) could be time-dependently dis placed by lanthanum in a Ca 21 -free, La 31 solution. Marshall and Kroeger (9) found an increase in tissue calcium content as measured by the lanthanum technique during K con tracture and a decrease when papaverine relaxed the K contracture. Our results with regard to high-K+ and papaverine favored their observations.