The Response of Creatine Kinase Specific Activity in Rat Pituitary to Estrogenic Compounds and Vitamin D Less-Calcemic Analogs

We examined the response of rat female pituitary at different metabolic stages to treatments with estrogenic compounds and vitamin D analogs. Immature or ovariectomized (Ovx) female rats responded by increased creatine kinase specific activity (CK) to estradiol-17β (E2), genistein (G), daidzein (D), biochainin A (BA), quecertin (Qu), carboxy- G (cG), carboxy- BA (cBA), and raloxifene (Ral). The response was inhibited when Ral was injected together with the estrogens. CK was increased when hormones were injected daily into Ovx rats for 4 different time periods. Pretreatment with the less-calcemic vitamin D analogs JK 1624 F2-2 (JKF) or QW 1624 F2-2 (QW) followed by estrogenic injection resulted in increased response and sensitivity to E2 and loss of inhibition of E2 by Ral. CK was also increased by feeding with E2 or licorice or its components dose- and time- dependent in immature or Ovxrats. Diabetic female rats did not respond to increased doses of E2. In conclusion, rat female pituitary is estrogens-responsive organ, suggesting to considerits response for HRT in postmenopausal women for both beneficial and hazardous aspects.


Introduction
Estradiol-17β (E 2 ) is essential for all aspects of reproductive function in females through activation of estrogen receptors (ERs). In the rat E 2 stimulates basal secretion of pituitary reproductive hormones [1]. The pituitary expresses both estrogen receptors, ERα and ERβ [2], and it responds to both ERα specific and ERβ specific agonists [33]. E 2 was found to have pleiotropic effects on physiological function in rat pituitary [3].
Phytoestrogens are plant-based estrogenic compounds, which are selective estrogen receptor modulators (SERMs) due to their ability to induce both agonistic and antagonistic effects. There is a growing interest in the use of phytoestrogens in Western countries. Widely marketed as food additives and present at fairly high concentrations in soy products [4,5], phytoestrogens are commonly treated in the lay media as a uniform class of naturally occurring estrogenic compounds retaining the beneficial effects of estrogens but carrying none of the harms potentially inflicted by native or synthetic estrogens. However, phytoestrogens vary considerably in terms of structure, estrogenic potency, and availability in common food sources such as soybeans, cereals, and sprouts [5]. Most human dietary sources contain phytoestrogens of two major chemical classes, isoflavones and lignans. The isoflavone genistein (G) is perhaps the best studied phytoestrogen [5][6][7] whereas data on the biological effects of other common isoflavones such as daidzein (D) or its metabolite equol [5][6][7] are relatively scarce. Based on favorable effects of these compounds on lipid oxidation [8,9] and vascular reactivity [5], a recent review of literature suggested that dietary phytoestrogen consumption may confer cardiovascular protection [6]. Phytoestrogens in the diet were also found to affect pituitary of rats, by modulating serum gonadotropin levels [10] similar to E 2 .
Licorice root extract (L) and its major isoflavans, glabridin (Gla) and glabrene (Glb), exhibited varying degrees of ERs' agonism in different tissues in vivo. Animals fed 2 International Journal of Cell Biology with L, Gla, and Glb similar to E 2 showed increased CK in different organs [11,12]. Phytoestrogens fed to rats were also found to alter some neurobehavioral effects [13] similar to E 2 .
Vitamin D binding protein is expressed in rat hypothalamus which shows its biological activity [14]. Vitamin D receptors (VDR) are also expressed in the hypothalamus [15] as well as in human pituitary [16] and in rat pituitary [17][18][19]. E 2 regulates a spectrum of activities in the pituitary as well as induction of calbindin D9K in the rat [20] which is also regulated by vitamin D metabolite 1,25(OH) 2 D 3 [21].
We have previously studied the hormonal modulations in different systems of the specific activity of the "estrogeninduced protein" creatine kinase BB [22], a rapid estrogen response-marker.
The present study was undertaken to see if, due to the presence of both E 2 and vitamin D receptors, the pituitary of female rats responds by induction of CK, to different hormones such as E 2 and different phytoestrogens with and without pretreatment with the less-calcemic vitamin D analogs JKF and QW by different ways of application and at different physiological status. The obtained results might suggest considering also the response of the pituitary to hormonal treatment, such as hormone replacement therapy (HRT) for postmenopausal women, is used for both its beneficial and its hazardous aspects.

Materials and Methods
Rats. (1) Wistar-derived prepubertal female rats, aged 25 days weighing 60 g at the start of the experiment (intact) or 2 weeks postovariectomy (Ovx), were used. Intact or Ovx rats were injected daily (5 days per week) for 10 weeks with either E 2 (5 μg/rat), Ral, G, cG, cBA, BA, or D (all at 500 μg/rat), or raloxifene (Ral 500 μg/rat) or all hormones with Ral, or all pytoestrogens together with E 2 . In other experiments, licorice (L, 25 μg/rat), or its synthetic derivatives glabridine (Gla, 25 μg/rat) or glabrene (Glb 25 μg/rat) with and without E 2 or Ral was given to rats by feeding for different time periods. The doses used were found in previous studies, to be the optimal effective doses in this model.
(2) Sprague Dawley female rats, at the age of 5 weeks, weighing 120 g, was injected subcutaneously with a single dose of Streptozotocin (STZ; 60 mg/Kg BW in 0.05 M citrate buffer, pH 5.7). Additional group of animals were injected with the vehicle (0.05 M citrate buffer, pH 5.7) and served as healthy controls. The animals were kept for 8 weeks in cages with 12 hours cycles of light and dark, Purina chow and tap water supplemented ad libidum [23]. Rats were used either as intact or 4 weeks postovariectomy (Ovx). E 2 at different doses was injected for 24 hours, followed by harvesting the pituitary for creatine kinase specific activity (CK) assay.
(3) For pretreatment with vitamin D less-calcemic analogs, Wistar-derived rats were used at initial age of 25 days. Female rats were used either as intact or after ovariectomy (Ovx), and treatments started 2 weeks postsurgery. Rats were injected daily for different time periods as indicated  with the analogs JKF 1624F 2 -2 (JKF) or QW 1624F 2 -2 (QW) (0.2 ng/gr BW), and 24 hours after the last injection, rats were injected with E 2 (0.5 μg/rat), raloxifene (Ral) (500 μg/rat) or both followed by harvesting the pituitary for creatine kinase specific activity (CK), assay.
Creatine Kinase Specific Activity Preparation and Assay. Rat pituitary was collected and homogenized in cold isotonic extraction buffer using a Polytron homogenizer. Enzyme extracts were obtained by centrifugation of homogenates at 14000 × g for 5 minutes at 4 • C. CK specific activity was measured in a Kontron Model 922 Uvicon Spectrophotometer using a Sigma coupled assay kit, and protein was assayed by Coomassie brilliant blue dye binding. Results are means ± SEM and are expressed as % of control of CK in hormonetreated compared to vehicle-treated, control animals.
International Journal of Cell Biology Statistical Analysis. Data were calculated as % stimulation by the treatment relative to control rats for each experiment as previously described. Comparison between the control and various treatments was made by analysis of variance using ANOVA.

Effects of Different Estrogenic Compounds on CK Specific
Activity in Immature and in Ovx Female Rats. When intact female (a) or Ovx (b) female rats were injected for 24 hours with either E 2 (5 μg/rat), genistein (G), carboxy-G (cG), biochainin A (BA), carboxy-BA (cBA) or daidzein (D) (all at 500 μg/rat), glabridin (Gla) (25 μg/rat), or glabrene (Glb) (25 μg/rat). All hormones that induced CK for different extent in pituitary from both type of animals ( Figure 1). Of note is the fact that most of the hormones were more active in pituitary from immature rats than in organs from Ovx rats.  Figure 3: Stimulation of creatine kinase (CK) specific activity by different estrogenic compounds with and without E 2 in pituitary from immature (a) and from ovariectomized female rats (Ovx; b). Rats were treated and assayed for CK activity as described in Section 2. Results are means ± SEM for n = 5-15 rats/group. Experimental means compared to control means: * P < .05 and * * P < .01. Basal activity in organs from immature and OVX rats were 1.02+0.15 μmol/min/mg protein and 0.67+0.22 μmol/min/mg protein, respectively.

Effects of Different Estrogenic
at 500 μg/rat), glabridin (Gla) (25 μg/rat) or glabrene (Glb) (25 μg/rat) or raloxifene (Ral 500 μg/rat) or all hormones with Ral. All hormones induced CK for different extent and enzyme induction in the pituitary by all compounds except Glb were inhibited when Ral was injected together with them ( Figure 2).

Effects of Different Estrogenic Compounds with and without
Estradiol-17β on CK Specific Activity in Immature and in Ovx Female Rats. Intact or Ovx female rats were injected for 24 hours with either E 2 (5 μg/rat), genistein (G), carboxy-G (cG), biochainin A (BA), carboxy-BA (cBA) or daidzein (D) (all at 500 μg/rat), or all hormones with E 2 . All hormones tested induced CK for different extent, but only cG and cBA when injected together with E 2 inhibited CK induced by each of them alone or E 2 alone (Figure 3).

Effects of Different Estrogenic Compunds Injected Daily for 4 Months on CK Specific Activity in Ovx Female Rats.
Ovx female rats were injected daily for 4 months with either E 2 (5 μg/rat), genistein (G), biochainin A (BA), carboxy-BA (cBA) or daidzein (D) (all at 500 μg/rat), or raloxifene (Ral 500 μg/rat). All hormones tested induced CK for different extents (Figure 4). Maximal stimulation was obtained by [Licorice] (μg/rat) Figure 5: Dose-dependent stimulation of creatine kinase (CK) specific activity by licorice, fed daily for 3 days, in pituitary from immature female rats, compared to feeding with single dose of E 2 . Rats were treated and assayed for CK activity as described in Section 2. Results are means ± SEM for n = 5 rats/group. Experimental means compared to control means: * P < .05 and * * P < .01. Basal activity was 1.05 + 0.20 μmol/min/mg protein.
E 2 whereas all other phytoestrogens had lower but similar effects.

Effects of Licorice Fed into Immature Female Rats at Different
Doses on CK Specific Activity. When intact immature female rats were fed daily for 3 days with either E 2 (5μg/rat) or licorice (L) at different doses (25-200 μg/rat), the compounds induced CK dose-dependently ( Figure 5). The daily  Figure 6: Stimulation of creatine kinase (CK) specific activity by licorice (L), glabridin (Gla) with and without E 2 in pituitary from immature (a) or from Ovx female rats (b). Rats were fed for different time periods as indicated and assayed for CK activity as described in Section 2. Results are means ± SEM for n = 5-10 rats/group. Experimental means compared to control means: * P < .05 and * * P < .01. Basal activity in Pi from immature rats was 0.97 + 0.22 μmol/min/mg protein and from Ovx rats was 0.67 + 0.18 μmol/min/mg protein.
feeding for 3 days with L was maximal at around 100 μg/rat, and this was similar to the stimulation by E 2 at 5 μg/rat.

Effects of Licorice or Glabridin or E 2 Injected into Immature or
Ovx Female Rats on CK Specific Activity. When intact or Ovx female rats were injected with either E 2 (5 μg/rat), glabridin (Gla 25 μg/rat) or licorice (L 25 μg/rat), or E 2 together with Gla or L, for different time periods, there was an increase of CK by all hormonal combinations injected, with no additivity with the combined treatments at both animal types ( Figure 6). In both animal types most of the effects were time-dependent with increased response at the longer time period measured. When Ovx female rats were injected daily for 4 weeks with either E 2 (5 μg/rat), glabrene (Glb 25 μg/rat) or E 2 together with Glb, all hormones induced CK for different extent, and when injected together with E 2 , CK induction by E 2 was inhibited by Glb (data not shown).  was induced for different extents only in intact rats but not in diabetic rats [23]. The response to E 2 which was not induced in the diabetic status was not seen even when higher doses up to 50 μg/rat of E 2 were used (Figure 7).

Effects of Estradiol-17β with and without Vitamin D Less-Calcemic Analogs on CK Specific Activity in Immature Female
Rats. When intact female rats were injected for 24 hours with either E 2 (at 0.5 μg/rat or 5 μg/rat) alone or with the less-calcemic vitamin D analogs JKF or QW (0.2 ng/gr BW) or daily for 3 days with the analogs followed by E 2 for 24 hours, all hormones induced CK when injected alone; after pretreatment with JKF or QW the response to E 2 was up-regulated by about 50% (Figure 8). When rats were injected with E 2 at 0.5 μg/rat, CK was up-regulated to even higher extent, indicating not only up-regulation of the response to E 2 but also increased sensitivity (Figure 8).

Effects of Estradio-17β with and without Vitamin D Less-Calcemic Analogs for Different Time Periods on CK Specific
Activity in Ovx Female Rats. When Ovx female rats were injected daily for 3 days, 1 week or 10 weeks with the lesscalcemic vitamin D analogs JKF or QW (0.2 ng/gr BW) and then E 2 (5 μg/rat) for 24 hours, CK was induced for different extent by the different hormones. The response of the E 2 was up-regulated by about 2 folds (Figure 9). The highest upregulation was noticed already after pretreatment for 3 days.

Effects of Estradiol-17β Together with Raloxifene with and without Vitamin D Less-Calcemic Analogs on CK Specific
Activity in Immature or Ovx Female Rats. Intact female rats were injected for 24 hours with either E 2 (5 μg/rat) or Ral  Figure 8: The effect of pretreatment with JKF and QW at 0.2 ng/rat for 24 hours (a) or for 3 days (b) on CK activity in Pi from immature female rats compared to the stimulation of E 2 at 0.5 μg/rat or 5 μg/rat and to the combination of the vitamin D analogs with E 2 . Treatments were as described in Section 2. Results are expressed as the ratios between the specific activities of CK in hormonetreated and vehicle-injected control animals. The basal activity of CK in was 0.86 + 0.14 μmol/min/mg protein after 24 hours and 0.92 + 0.18 μmol/min/mg protein after 3 days, n = 5. * P < .05, * * P < .01 in the difference compared to vehicle.
(500 μg/rat), or E 2 and Ral alone, or after daily injections for 3 days with the less-calcemic vitamin D analogs JKF or QW (0.2 ng/gr BW). All hormones induced CK when injected alone, but when E 2 was injected together with Ral, CK induced by E 2 was inhibited. After pretreatment with JKF or QW by daily injections for 3 days followed by E 2 , Ral, or E 2 + Ral, the response to E 2 but not to Ral was upregulated by about 50%, but when injected together there was no more inhibition of E 2 by Ral (Figure 10(a)). When Ovx female rats were pretreated similarly but for 1 week instead of 3 days, similar results were obtained, but at higher extent ( Figure 10(b)).

Discussion
The key finding in the present study is that rat female pituitary is a hormone-responsive organ which responds at different stages of development, to different estrogenic compounds similar to other rat organs such as the skeleton, the uterus, and the vascular ones [29]. First, E 2 as well as different phytoestrogens and their carboxy-derivatives stimulate CK activity in rat pituitary of both immature and Ovx similar to other estrogen-responsive organs such as the skeleton and the vasculature ones, both by single and long-term multiple injections [29]. Second, E 2 as well as different phytoestrogens and their carboxy-derivatives stimulate CK activity in rat pituitary of both immature and Ovx similar to other estrogen-responsive organs such as the skeleton and the vasculature. This response is inhibited by the SERM Ral [29][30][31]. Third, all phytoestrogens-induced CK were not significantly affected by addition of E 2 , except that of cG and cBA behaved like SERMs and inhibited E 2 stimulated CK similar to other estrogen-responsive organs such as the skeleton and the vasculature [32]. Fourth, the less-calcemic analogs of vitamin D, JKF and QW, per se increased CK in rat pituitary; moreover, pretreatment with JKF or QW for different time periods at both immature and Ovx rats up-regulated the response and the sensitivity to E 2 similar to other estrogen-responsive organs such as the skeleton and the vasculature [25,30]. Fifth, licorice and the phytoestrogens derived from it, that is, glabridin and glabrene also stimulated rat pituitary CK activity from both immature and Ovx rats, when applied for different time periods either by feeding or injections. This stimulation was dose dependent. It is of interest to note that unlike Gla, Glb was also SERM-like and inhibited E 2 stimulated CK similar  Figure 10: The effect of pretreatment with JKF and QW at 0.2 ng/g/rat/day for 3 days in immature female rats (a) or 1 week in Ovx female rats (lower panel) on CK activity compared to the stimulation by E 2 at 5 μg/rat or Ral at 500 μg/rat or both, and to the combination of the vitamin D analogs with E 2 , Ral, or both. Treatments were as described in Section 2. Results are expressed as the ratios between the specific activities of CK in hormonetreated and vehicle-injected control animals. The basal activity of CK in was 0.78 + 0.10 μmol/min/mg protein for Ovx and 1.02 + 0.12 μmol/min/mg protein for immature female rats, n = 5. * P < .05, * * P < .01, in the difference compared to vehicle.
to other estrogen-responsive organs such as the skeleton and the vasculature [31]. Sixth, E 2 failed to stimulate CK activity in pituitary from diabetic immature and Ovx rats, even at increased doses, similar to other estrogen-responsive organs such as the skeleton and the vasculature [23]. Previous studies showed that the stimulation of CK in the pituitary is similar to other rat organs, such as epiphysis and diapyhsis in the skeleton as well as aorta and left ventricule in the vasculature, but not in the uterus [30].
This is in accordance with other studies clearly demonstrating that the pituitary is influenced by E 2 in both immature and Ovx rats, when other biological responses such as hormonal secretion were determined [3]. This is similar to other studies demonstrating the presence of ERs in the rat pituitary, which are responding to E 2 as well as to phytoestrogens like revestranol [3]. Moreover phytoestrogens were found to influence also brain development, neural function, and behavior parametrs [13].
The licorice derived phytoestrogens were previously shown by us to have similar effects on the skeleton and the vasculature both in vivo and in vitro in cell derived systems [11,12,27,31].
The dietary estrogens exert biological activity and affect gonadotropin release from the pituitary [10] via both ERα and ERβ present in the rat pituitary [33], but the present 7 study is showing directly modulation of CK, which is an estrogenic marker, in rat pituitary.
Vitamin D active metabolites have important physiological effects which are mediated via intracellular receptors which are present in a variety of organs and tissues including rat and human pituitary [34,35]. Vitamin D also affects hormone secretions in the rat pituitary [36]. Moreover, E 2 regulates vitamin D-mediated calcium absorption by the induction of cytosolic calcium binding protein (CaBP-D9K) [37] via ERs in the rat pituitary [38].
As previously shown in other organs [30], vitamin D analogs up-regulate the response and the sensitivity of different rat organs from both immature and Ovx rats to E 2 and to different phytoestrogens. In the present study, the pituitary was shown to have similar properties, suggesting that there is mutual modulation of this hormonal responsiveness in this organ as well.
We have noticed that in diabetic rats the response to E 2 of different rat organs from both immature and Ovx rats was abolished compared to intact rats [23]. In the present study, the pituitary was shown to have similar properties, namely, complete abolition of estrogenic response of rat pituitary in the diabetic condition at both types of rats, even when increased hormone levels were injected.
In conclusion, rat female pituitary is a hormoneresponsive organ which responds differently at different stages of development, to a variety of estrogenic compounds similar to other rat organs such as the skeleton and the vasculature but not the uterus. It is important to notice that all compounds were effective in using them at their optimal dose. But in immature female rats E 2 and G were most effective, and this small difference was not apparent in Ovx female rats. Moreover it also responds to vitamin D analogs alone and to their up-regulation properties. These might suggest taking into consideration the response of the pituitary in addition to the other organs, when hormonal treatment, such as hormone replacement therapy for postmenopausal women, is used for both its beneficial and hazardous aspects.