Effects of tamoxifen citrate and cycloheximide on

Effects of tamoxifen citrate and cycloheximide on estradiol induction of rat myometrial gap junctions' Departments of~eurosciencesand Obstetrics and Gynecology, McMasfer University Health Sciences Centre, Hamilton, Ont., Canada L8N 325

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Received August 16, 1985

MACKENZIE,L. W., and R. E. GARFIELD.1986. Effects of tamoxifen citrate and cycloheximide on estradioI induction of rat myometrid gap junctions. Can. J. Physiol. P h m a c o l . 64: 703-906. Longitudinal muscle of myometrial tissues from immature rats were examined by quantitative thin section electron microscopy for the presence of gap junctions after treatment with estradiol with and without tamoxifen, and cycloheximide for 1-6 days. Gap junctions were present between myornetrial cells on days 4, 5, and 6 after treatment with estradioI (580 pglday). Tarnoxifen administered concornitmtly with estradisl over the 6-day period completely prevented induction of the junctions. Gap junctions were not detected in the myometrium after treatment with tamoxifen alone. Adrrministxation of cycloheximide together with estradiol on day 0 of the 6-day period had no effect on gapjunction frequency but resulted in a reduction in gap junction size in the myomtrium after continued treatment with the hormone. Treatment with cycloheximide on day 1, however, significantly suppressed the effect of further estradiol treatment on the induction of gap junctions in the myometrium. Junctions were not visible in the tissues from animals treated with cycloheximide alone or in the control groups treated with sesame oil. These results indicate that estradioI influences the presence of gap junctions in the myometrium by regulating the synthesis of gap junction proteins though the steroid receptor mechanism. MACKENZIE,L. W., et R. E. G A R ~ E E B 1986. . Effects of tamoxifen citrate and cycloheximide on estradiol induction of rat myornetrial gap junctions. Can. 9. Physiol. P h m a c o l . 64: 703-706. On a examine le muscle longitudinal de tissus de myom2tre de rats immatures par microscopic Clectronique quantitative de couches fines afin de dktenniner la prdsence de jonctions lacunaires aprh un traitement avec de l'oestradiol avec et sans tamoxifen, ainsi qu9avecde cycloheximide et ce, pendant 8-6 jours. On observa la prdsence de jonctions lacunaires dans Bes celldes du rnyomhtre aux jows 4, 5 et 6 apr&s le traitement avec 190estradiol (508 pg/jsur). Le tamoxifen administrk conjointement avec I'oestradiol pendant une pkriode de 6 prkvint totalement I'induction des jonctions. On ne dCtecta pas de jonctions lacunaires dans le myom8tre apr8s le traitement avec le tamoxifen uniquement. L'administration de cycloheximide en m6me temps que celle dbestradiol le jour0de la pCriode de 6 jows n'eut pas d'effet sur la frbquence des jonctions lacunaires mais rCsulta en une rkduetisn de la dimension des jonctions lacunaires dans le myomktre aprks un traitement continu avec I'homone. Toutefois, un traitement avec le cyclohexirnide le jour 1 supphima efficacement l'effet d'un autre traitement avec l'oestradiol sur l'induction de jonctions lacunaires dans le myomktre. Les jonctions n'ktaient pas visibles dans les tfssus des animaux trait& avec le cycloheximide seul ni chez Ies groupe tCmoins trait& avec de l'huile de sCsame. Ces rCsultats indiquent que l'oes$rtfdiol influence la pdsence de jonctions lacunaires dans le rnyodtre en regularisant la synthkse des protkines des jonctions lacunaires par le mkcanisme rkcepteur des stCroides. [Traduit par la revue]

ours

Introduction Recently we have shown that large doses of estradiol stimulate the in vfvo development of gap junctions in immature and ovouiectomized mature rats (MacKenzie and Garfield 1985). Gap junctions are aggregates of proteins within the plasma membrane (Beracchia 1980) and it is well known that estradiol regulates protein synthesis in the uterus (Gorski et al. 1968). It is possible, therefore, that estradiol controls the presence of myornetrid gap junctions by influencing protein synthesis. It has been proposed that de novo protein synthesis may be necessary for gap junction formation in myometrial tissues incubated in vita0 (Garfield et al. 1980). Moreover, a rnRNA preparation extracted from the myornetrium of rats after treatment with estrogen and then incorporated into a gap junction deficient cell line has been suggested to bring about the development of gap junctions between those cells (D&l et al. 1980). Thus, these studies provide circumstantial evidence for a direct action of estrogen on protein synthesis in regulation of rnyometrial gap junctions. In addition, it has also been reported from studies using cycloheximide to inhibit the formation of gap junction between other cell types, that de novo protein synthesis is required for the expression of the junctions (Decker et al. 1978). uppo ported by Medical Research Council of Canada grants to Dr. R. E. G d e l d . '~uthorto whom all correspondence may be addressed.

Some studies have indicated, however, that de novo protein synthesis may not be required for gap junction formation in all cell types (Epstein et al. 1977; Kannan and Daniel 1978). It is generally thought that estradiol regulation of uterine protein synthesis occurs through the well-known classical steroidreceptor mechanism (GBasser et al. 1977). It has been shown that estradiol stimulation of W A and protein synthesis is suppressed by the antiestrogen tamoxifen though the steroid receptors (Koseki et al. 1977). In this study we have evaluated whether de nsvo protein synthesis is required for estradiol stimulation of myometrial gap junctions in vivs and if the steroid receptor mechanism is involved in the process

Materials and methods: Animals Immature female Wistar rats (21-30 days old, weighing 30-60 g) were used throughout the experiments. The animals were housed four to five per cage, maintained at a constant temperature (22°C) with a photoperiod of 12 h light, and were fed hhina rat chow and water ad libitum. Treatmend Animals were injected subcutaneously with estradiol-17P (500 pg/ day in 0.1 mL sesame oil) with and without tamoxifen citrate suspended (5 mg/O. 1 mL) in sesame oil or cyclohexidde dissolved (100 pg/O.l d ) in physiological saline. Control animals were treated with sesame oil alone. The injections were started on the morning of the

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CAN. I. PHYSIOL. PH

2 2 d day s f age and were repeated every 224 h for 1-6 days as indicated for ~e paticdaf experiment. Exactly 24 R after the required number of injections the rats were killed by cervical dislocation following a blow to the head. Samples s f uterine dssue were immediately removed and processed for analysis by thin section eIectrsn microscopy. Electron ~ C C ~ O S C O J P P ? Tissue collection, processing for electron microscopy and method of mophornetric quantitation of gap junctions in longitudinal muscle have k e n described in detail elsewhere (MacKenzie and Garfield

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1985). Statistical comparisons

The Mm-Whitmy U-test was used for statistical comparisons on quantitative measurements of gap junctions.

Results Efects 6P$garn~xfen To mdyse the effects of an antiestrogen on the presence of myornetrial gap junctions in the immature rats, tamoxifen (5 mdday) was administered done or together with 500 pglday estradiol for 1-6 days. Gap junctirssns were absent or present at an extremely low frequency in tissues from control animals a d rats treated with tmoxifem done over the 1-61 day period (Fig. 1A). Similaply, the frequency of myornetrial gap junctions was low over the first 3 days of treatment with 500 pglday esbradisl. Thereafter, however, the frequency was significantly elevated (P < 0.05) after 4 , 5 and 6 days of treatment. When tamoxifen was injected simultaneously with estradiol over the 1-6 day period, gap junctions were absent s r present at a very low frequency compared with estradiol treatment dome m d similar to that of control md tarnoxifen-treated animals. The sizes of gap junctions in tissues from control md tmoxifen-treated animals after four md six injections were very small (40and 60 nm, respectively, Fig. 1B). The mean size of gap junctions stimulated by estradiol increased to a near maximum size after 2 days of treatment which was maintained with repeated injections over the remainder of the 6-day period. In myornetrid tissue from mirnds treated with estradiol plus tamoxifen, the gap junctions observed after 2 and 6 days of treatment measured 160 and 30 nm,respectively. The percentage of myornetrid membrane area occupied by gap junctions in tissues from control, tarnoxifew, and estradiol plus tamoxifen-treated animals over the 1-6 day period and in a n i d s treated with eskadiol for 1-3 days was extremely low (Fig. 1C). The gap junctional membrane area after 4 days of estradiol treatment was significantly larger (P < 0.05) compared with h e controls treated with sesame oil alone. The increase in membrane area of gap junctions after estradiol treatment was maintained with repeated injections and was significantly greater (P < 0.01)after 6 days of treatment compared with the membrane area in control, tamoxifen, or estradiol a d tamoxifen-treated tissues. E.ects sf cyc&oheximide The effects of cycloheximide on the presence of myornetrial ature rats are shown in Table 1. Cyclohexh i d e (100 pg) was administered alone or together with estradiol(5gBbd vg/day) at zero time (day 0)or on day 1 following the start of estradisl treatment. UltrastmctmH analyses for myornetrid gap junctions were carried out after 4 and 5 days of treatment with estradiol. Gap junctions were present at an extremely low frequency and small size, occupying a small percentage of the myometkal membrane area in tissues from con&o1 animals treated with sesame oil. Gap junctions were not

2

3

4

DAYS

FIG.1 . Effects of tarnoxifen on myornetrial gap junctions (GJ). Tmoxifen was administered alone or together withesttadiol daily over 1-6 days to determine its effects on estradisl-induced gap junction fornation. (A) Number GJI1CY.H pm, frequency of gap junctions per 1W prn measured membrane length; (B) size, length (nm) of each gap junction; ( C )m a of GJlwea plasma membrane, m a of gapjunctions is expressed as percentage s f gap junction area per area plasma membrane. Frequency, size, and area of gap junctions are given as mean values f SE of four to six tissues (equal to number of animals) for treatment on each day. Numbers in parentheses (B) represent the total number of gap junctions found on that specified day. observed in the myometrium from rats after 4 or 5 days following treatment with cycloheximide alone on day 0 or day 1 . The concomitant administration of cycloheximide (180 pg) with estradisl (500 ~ g l d a y )to immature rats on day 0 had no influence on the frequency or membrane area of gap junctions in the rnyometrium after a further 4 or 5 days of treatment with estradiol compmd with values from animals treated with estradid alone over the same perids (Table 1). The mean size of the junctions in tissues from estradiol-treated animals that received an injection of cyclohexirnide on day 0,however, were smaller than those in tissues from rats treated with estradiol alone. When animals injected with estradiol were treated with a simultaneousdose of cycloheximide on day 1,24 h after the first day of treatment with estradiolj, there was a significant suppression in the frequency (P< 0.0 % and B 4 0.05) and percentage (P < 0.01) sf membrane area occupied by gap junctions after coasthued treatment with estradiol over 4 and 5 days compared with the estradioB treatment alone (Table 1). After 4 and 5 days of treatment the mean gap junction size was smaller in the group of animals treated with cycloheximide on day 2 with a significant difference (Ha< 0.05) occurring after 4 days.

Discussicsn This study shows the effect of tamoxifen, a synthetic nsnsteroidd antiestrogen, on estradiol-induced myornetrial gap junc-

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TABLE1 . Effect of estradiol (E2)and (or) cycloheximnide (Cyclo) on myometrial gap junctions (GJ) in immature rats Length of

No. of

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Treatment

tissues

No. sf GJs

membrane

No. of GJs/lHlQ bm surveyed (pm) membrane

Mean size GJs (nm)

Area GJs/ma plasma membrane

(%I

4 days Control Cyclo (day 0) Cyclo (day 1) 5 2 (580 F B / ~ ~ Y ) E2 plus Cyclo (day 0) E2 plus e y c b (day 1 ) 5 days Control Cycle (day 0) Cyclo (day 1) E2 (508 pi3'day) E2 plus Cyclo (day 0) E2 plus Cyclo (day 1) NOTE:Immature rats were injected daily for 4 and 5 days with estradiol(580 geg) with and without an injection of cycloheximide (100 gkg) on day 0 (zerotime) or day 1 to determine the effect of protein synthesis on myonnetrial gapjunctions.No. of tissues, total number of tissues examined;No, of GJs, total number of gapjunctions found within 20-22 wnoverlapging photowkrograplksof each tissue; No. of GJs11008 pm membrane, frequency of gapjunctions per IOOO pm measured membrane length; size d GJs, bmg&of each gap junction; area of GJs/m;aof plasma rnembme, area of gapjunctions expressed as a percentage of gapjunction area per area plasma membrane. Frequency. x not significantlydifferent(P> 0.05). size, and m a of gap junctions a e given as mean values + SE.Mean values with the same superscfiptsa

eions. Tamoxifen did not promote the induction of myometrial gap junctions in the immature rat over a 1-6 day period. Moreover, tamoxifen was found to completely suppress the ability of estradiol to stimulate the presence of gap junctions in the myometfium. It is generally thought that tamoxifen acts as a partial agonist-antagonist to estrogenic uterine responses (Jordan and Dix 1979). With respect to myometrial gapjunctions, however, tamoxifen seems to act as a pure estrogen antagonist. Observations from a previous study based on semiquantitative rnorphometq (Bwghxdt et al. 1984) support the antagonistic effect of tamoxifen on estradiol stimulation of myometrial gap junctions in nonpregnant rats. Tamoxifen exerts its antiestrogenic effect in the rat uterus by binding to the cytoplasmic estradiol receptor followed by the trarnsltxation of the antiestrogen-receptor complex into the nucleus of the cell (Katzenellenbogen et al. 1978). The mechanism by which the tmoxifen-receptor complex inhibits estradiolinduced uterine responses, however, has not been resolved but appears to result from altering the steroid receptor mechanism. Koseki et al. (1977) have shown that the tamoxifen-receptor complex is retained in the nucleus for a much longer period of time than estradiol. This observation suggests that the inability of the cell to process or remove the tamoxifen-receptor complex rapidly from the nucleus, as it does with estradiol, might somehow interfere with continued uterine stimulation. Prolonged nuclear retention of the antiestrogen-receptor complex and the biological action characteristic of the antiestrogen may be the result of the complex binding to different nuclear loci or sites inaccessible to estradiol resulting in loss of estrogenic responses (Sutherland and Murphy 1982). The antiestrogenic effects of tamoxifen therefore, implies that estradiol regulates the presence of gap junctions in nonpregnant myometfium though the steroid receptor mechanism. Gapjunctions are also present in the myometrium of pregnant animals immediately prior to and during tern or pretem labour and the change in levels of the steroid hormones are thought to be involved in regulating their presence and labour (Garfield 1984). It is highly likely that the steroid receptors regulate gap

junction development in the pregnant myometrium as they do in the nonpregnant state as shown in this study. Studies of pregnant animals treated with tamoxifen may provide significant insights into myornetrid gap junctions and parturition. The present study also reveals that de novo protein synthesis may be necessary for the presence of gap junctions in the immature rat myometrium in response to estradiol. Inhibition of protein synthesis by cycloheximide prevented the increase in frequency, size, and membrane area of gap junctions in myornetrid tissues from animals treated with esepdiol (Table 1). Although this study does not provide evidence showing that cycloheximide reduced protein synthesis in the immature uterus, it has previously been reported that cycloheximide at a dose similar to that employed in this study blocks the incoagoration of [14~]leucineinto trichloroacetic acid precipitable protein in the immature (Sariff m d Gorski 1971), and the intact and ovariectomized mature rat (Cidlowski and Muldoon 1978). Furthermore, cycloheximide has also been shown to prevent uterine responses to estrogen, such as estrogen-induced myornetrial contractions and steroid hormone receptor synthesis (Rexoad 1978; Dix and Jordan 1988). In addition, it has been reported that cycloheximide inhibits increased numbers and sizes of gap junctions between other cells (Decker et al. 1978) and in the mysmetrium studied in vitao (Garfield et al. 1988). The results of this study indicate that if protein translation is blocked, estradiol treatment cannot induce the formation of myornetrial gap junctions. This is suggestive that estmdiol stimulation may result in the transcription of a specific proteins which f o m the molecular structure of the gap junctions. Dahl et al. (1980) have reported that a &NA preparation extracted from the myometriurn of estrogen-treated rats induced gap junction formation in a cell line deficient in the junctions. The above study, however, did not specify the dose or estrogen used, the number of treatments the animals received, or when the mRNA was extracted after commencing the treatment. The data from the present investigation indicate that these are important parameters to consider. The study of Bahl et ale (l98Q), however, does suggest that HBE9A and protein synthesis may be

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CAN. J. PHYSIOL. PHAHBMACOL. VOL. 64, 1986

necessary for the expression of gap junctions. It is evident, therefore, that estradiol stimulates the synthesis of gap junction proteins through the receptor mwhanisrn. It appears also that the prostaglandins we involved in the fomation of myornetrial gap junctions by regulating the effect of steroid hormones via decreasing the number or binding capacity of their receptors (Mackenzie et al. 1983; MacKenzie and Garfield B 985). Concomitant administration of cyclohexirnide with estradiol at day 0 in this study failed to prevent the appearance of gap junctions in the myornetxiurn after continued homone treatment. We did not administer cycloheximide on a daily basis as with the estradiol treatments because of the toxicity of the drug. The inability of cycloheximide administration on day 0 to prevent the appearance of gap junctions after continued hormone treatment may indicate that a single injection of cycloheximide is not sufficient to completely block total protein synthesis in this model system. When cycloheximide was given on day 8, however, there was a reduction in the size of the junctions. This indicates that a mechanism dependent on protein synthesis may be necessary for assembly of connexons into gap junction stmctures. Cycloheximide, however, prevented the increase in myornetrial gap junctions when administered concomitantly with the second injection of estradiol 24 h after the first day of treatment. At present we do not understand why cycloheximHde suppressed gap junctions only when administered with estradiol om day S of the treatment period, but not when given simultmeously with estradiol at the beginning of the treatment period. This could be suggestive of a high concentration of connexons required for gap junction fomation and this level may not be attained whew protein synthesis is prevented on day 1 of the treatment period. Alternatively, a sufficient quantity of gap junction proteins may be synthesized after one treatment with estraciiol but that another system involved in estradiol stirnulation of gap junctions may be suppressed by administration of cycloheximide on day 1. These results further suggest that protein synthesis may be required for at least 48 h in response to repeated estradiol injections for the expression of gap junctions in the immature rat myometkiiurn. A recent observation that a single ira~edionof estradiol on day 0 failed to induce the presence of numerous myornetrid gap junctions over a 1-6 day period in the immature rat is supportive of the above hypothesis (LoW. MacKenzie and R. E. G h e l d , unpublished obsemations) . It has been reported that cyclohexirnide, at a concentration similar to that used in this study, reduces estpadiol-induced uterine protein synthesis by approximately 95% for about 6 h dter hormone administration (Sariff and Gorski 1971). One might have expected ahat both treatment schedules with cycloheximide would have depressed or prolonged the appearance of the junctions at 4 and 5 days. Since this was not the case, one can only conclude that many factors are probably involved in the synthesis and assembly of gap junctions dter estradiol treatment. A more detailed study is warranted. We attribute, however, the effect of cycloheximide as evidence that protein synthesis is required for the presence of the junctions in the rat myornetrium in response to treatment with estradiol.

B U R G H A R. ~ TC., , P. A. MITCHELL, and R. KURTEN. 1984. Gap junction modulation in rat uterus. II. Efects of antiestrogen on myornetrial and serosal cells. Biol. Reprod. 30: 249-255. CKDLOWSKI, J. A., md T. G. Mv~mow.1978. The dynamics of intercellular estrogen receptor regulation as influenced by 178estradiol. BioB. Regrod. 18: 234-236. DAHL,G., B. AZARNBA, and R.WERNER. 1980. De novo construction of cell-to-cell channels. In Vitro, 16: 1068- 1075. DECKER, R. S., S. T. DONTA, W. J. LAWSEN, md S. A. MURRAY. 1978. Gap junctions and ACTH sensitivity in Y-l adrenal tumor cells. J. Suprmol. Stmct. 9: 497-503. Dax, C. J., and V. C. Jomampa. 1980. Modulation of rat uterine steroid homone receptors by estrogen and mtiesterogen. Endocrinology (Baltimore), 107: 2Q11-2020. EPSTEIN, M. k., I. D.SHERIDAN, and W. G. JOHNSON. 1977. Fomakion of low resistance junctions in vitrs in the absence of protein synthesis and ATP production. Ewp. Cell Res. 1M: 25-30. GARFIELD, R. E. 8984. Control of myornetrial function in p ~ k m versus tern labor. In Clinical obstetrics and gynecology. Edited by I. R. Merkatz and W. R.Keye. Harper and Row Inc., New York. pp. 572-591. GARFIELD, R. E., D. M E ~ E T T and , A. K. GROVER. 1980. Gap junctions fomation and regulation in rnyometrium. Am. 5. Phy siol. 239:C217-C228. GEASSEW, S. R., B. H.CLARK, R. G. SMITH, and B. W. O'MALLEY. 1977. Mechanism of steroid hornme action. I n Endocrinology of pregnancy. Edited by F. Fuchs and A. Klooper. Haper and Row, New York. pp. 15-40. GORSKI, J . , D. Tom, G. SHYAMAEA, D. SMITH,and A. NOTIDES. 1968. H m o n e receptors: studies on the interaction of estrogen with the uterus. Recent h o g . Horn. Wes. 24: 45-80. JORDAN, V. @. , and G. J. Drx. 1979. Effects of estradiol benzoate, tmoxifen and rnonohycHroxytamoxifen on immature rat uterine progesterone receptor synthesis and endometrial cell division. J. Steroid Biochem. 11: 285-291. KANNAN, M. S., md E. E. DANIEL. 1978. Formation of gap unctions by treatment in vibro with po&ssium conductmce blockers. J. Cell Biol. 78: 338-348. KATZENELLENBOGEN, B. S., J. A. KATZENELLENB~EN, E. Ha. PERGUSON, and N. KRANTHAMEW. 1978. Antiestrogen interaction with uterine estrogen receptors. J. Biol. Chern. 253: 697-707. KOSEKI, Yo,D. T.ZAVA,G . C. CHAMNESS, and W. L. MCGUIRE. 1977. Estrogen receptor translocation and replenishment by the antiestrogen tamoxifen. Endocrinology (Baltimore), 101: 11048 118. MACKENZIE, L. W., and R . E. GARRELD. 1985. Hormonal control sf gap junctions in the rnyometPiurn. Am. B. Physiol. 248: C296C388. MACKENZIE, L. W., C. P. PURH, and W. E.GARFIELD. 1983. Effects of estradiol-17p and prostaglandins on rat myornetrial gap junctions. Prostaglandins,26: 925-94 1 . ~ R A C ~C.H 1988. ~ A Stmctufal , correlates of gap junction permeation. Int. Rev*Cyto1. 66: $1-146. REXROAD, C. E. 1878. Cycloheximide blockage of estrdiol-17p induced contractions in ovaiectomized ewes. Biol. Reprod. 19: 648652. SARIPF, M., md J'. GOWSKI. 1971. Conbro1 of estrogen binding protein concentration under basal conditions and after estrogen administration. Biochem. 10: 2557-2563. SUTHERLAND, R. L., and L. C. MURPHY. 1982. Mechanisms of estrogen antagonism by nonsteroidal antiestrogens. Mol. Cell. Endscrinol. 25: 5-23.