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Pii: s0304-8853(00)01262-2

Journal of Magnetism and Magnetic Materials 225 (2001) 226} 234 Signal transduction of erythrocytes after speci"c binding of ecdysterone and cholesterol immobilized on nanodispersed O.M. Mykhaylyk *, A.V. Kotzuruba
, O.M. Buchanevich
, A.M. Korduban, E.F. Meged
, N.M. Gulaya
Institute for Applied Problems in Physics and Biophysics, National Academy of Sciences, Ukraine, P.O. Box 355, 255001 Kyiv, Ukraine
Institute of Biochemistry, National Academy of Sciences, Ukraine, Leontovicha 9, 252030 Kyiv, Ukraine Institute of Physics of Metals, Vernadskogo 36, 03142 Kyiv, Ukraine Concurrent binding of cholesterol and ecdysterone immobilized on nanodispersed magnetite to intact rat erythrocytes was investigated. Several binding components on erythrocyte plasma membrane with di!erent a$nities were revealed inthe range of 10 }10  M. The speci"c binding modulates signal transduction through adenylate cyclase and guanylatecyclase systems as manifested by the decrease in cAMP and increase in cGMP second messenger production.  2001Elsevier Science B.V. All rights reserved.
Keywords: Nanodispersed magnetite; Ecdysterone; Cholesterol; Membrane e!ects; Signal transduction; Immobilization; cGMP;cAMP; Erythrocyte cellular signaling were shown to be associated withspeci"c membrane receptors completely di!erent Steroid hormones are being increasingly used for from those of intracellular receptors, which was correction of a set of pathological states in human supported by the inability of classic steroid recep- body. Genomic e!ects of steroids (induction of spe- tor antagonists to inhibit non-genomic steroid ac- ci"c genes expression as a result of binding with tions [2]. Steroids can produce non-genomic e!ects soluble hormone receptors) have been known for on cells, such as opening of ionic channels, mem- decades, and rapid actions of steroids have been brane receptor aggregation or changes in protein more widely recognized and characterized only re- cently [1]. Rapid e!ects of steroid hormones on The existence of insulin, adrenaline, noradrena- line, glucocorticoid receptors on the erythrocytesmembrane is well documented [3,4]. Steroid hor-mones are transported with erythrocytes [5]. Thequestion is under discussion whether diversity of * Corresponding author. Tel.: #380-44-252-57-71; fax: receptors that mediate signal transduction in E-mail address: [email protected] (O.M. Mykhaylyk).
non-erythroid cells may be vestiges of signaling 0304-8853/01/$ - see front matter  2001 Elsevier Science B.V. All rights reserved.
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O.M. Mykhaylyk et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 226} 234 Fig. 1. Chemical structure of 7-dehydrocholesterol (a), calcitriol (b) and ecdysterone (c).
pathways critical to functions of erythrocyte pre-cursors but no longer needed in the mature eryth-rocytes or they are involved in erythrocytefunctioning [6]. There are no data on the existenceof C-steroid hormone receptors on the eryth- rocyte plasma membrane.
The structure of the C-steroid ecdysterone is similar to that of calcitriol, because they are syn-thesized from the same precursor 7-dehydrocholes-terol (Fig. 1). Rapid non-genome action of calcitriolin various types of cells has been found [7]. Also,rapid signal e!ects of calcitriol and ecdysterone invitro and in vivo [8] may be transmitted by speci"cmembrane binding sites. Furthermore, experi-mental data suggest a possible role of free Fig. 2. Hypothetical scheme for molecular mechanisms of recep- cholesterol as lipid second messenger [9]. This is tor-mediated stimulation of cyclic GMP formation in eryth- supported by the fact that the blockin plasma rocytes. E, C-steroid; MC, magnetic carrier; R and R, membrane cholesterol transfer to intracellular receptors; AC, adenylate cyclase; pGC, particulate or membran-ous guanylate cyclase; sGC, soluble guanylate cyclase; Gs and membranes occurred at the level of the plasma Gi, G-protein stimulating and inhibiting subunits; NOS, NO- membrane and the onset of inhibition was rapid synthase; HO, heme oxygenase;PKC and reversible [10].
?;
-isoform of protein kinase C; cGMP, cyclic 3
,5
guanosine monophosphate; GTP, In Fig. 2, we propose a scheme for molecular guanosine triphosphate; cAMP, cyclic adenosine monophos- mechanisms of receptor-mediated stimulation of phate; ATP, adenosine triphosphate; NO, nitric oxide; IP, inositol 3,4,5-triphosphate; DAG, 1,2-diacylglycerol; PGE cyclic 3
,5
-guanosine monophosphate (cGMP) formation in erythrocytes. It is known that eryth- ; CO, carbon monoxide; Hb, hemoglobin; ROS, reactive oxygen species.
rocytes display membranous adenylate cyclase ac-tivity [11] and recently it was shown that humanerythrocytes possess not only soluble but alsomembranous or particulate guanylate cyclase [12].
synthesis of compounds speci"c for the phospolipid In our previous workusing ecdysterone immobi- pathway [13]. Activation of phospholipid hydroly- lized on nanodispersed magnetite, we have shown sis can lead to the increase of intracellular calcium the existence of high-a$nity ecdysterone-speci"c concentration which is stimulus for guanylate cyc- binding sites (K&1.7;10 ) on the surface of lase activation [14,15] via stimulation of NO-syn- erythrocytes and investigated the e!ects of 0.1 M thase. The stimulation of guanylate cyclase by ecdysterone on signal transduction of human eryth- arachidonic and other free fatty acids, eicosanoids, rocytes and revealed a metabolic response leading reactive oxygen species as well as by 1,2-diacyl- to phospholipase A activation and enhanced glycerol via protein kinase C? activation is well

O.M. Mykhaylyk et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 226} 234 established in numerous experimental systems magnetite are shown in Fig. 3. The mean diameter [16,17]. Direct activation of voltage-dependent of particle cores was approximately 25 nm. The Ca>-channels, membranous guanylate cyclase activation of particle surface was performed with and heme oxygenase I which produce a powerful 1,6-hexamethylendiisocyanate in a dry aprotonic guanylate cyclase activator (carbon monooxide) solvent (methylenchloride) using tetrabutyltitanate [18,19] is also possible in response to hor- as a catalyst, as described previously [21]. Ecdys- mone}receptor interaction. In the present work, terone was chemisorbed on the surface of the we addressed the question whether there are activated carrier from 2.5 mM acetone solution speci"c binding sites for cholesterol and ecdys- while cholesterol was chemisorbed from 2.8 mM terone on the erythrocyte plasma membrane in the solution in 0.05 M Na-phosphate bu!er, pH 7.2, 10 }10  M concentration range and whether then the adsorption of glycine from its 0.1 M solu- these binding sites perform receptor function and tion in 0.05 M Na-phosphate bu!er, pH 7.2, fol- modulate cAMP-dependent signal pathway and lowed in both cases. The activated magnetite with cGMP-dependent transduction followed on the chemisorbed glycine was used as a reference sample level of 3
,5
-cyclic adenosine monophosphate M. The concentration values of immobilized (cAMP) and cGMP second messengers.
ecdysterone (E) and cholesterol (Ch) moleculeswere estimated from the decrease in optical densityof ecdysterone (cholesterol) absorption in the 2. Experimental methods "242 nm (210 nm), "12 200(34 000) M  cm  [22] and were found The crystalline ecdysterone used was obtained to be 25 mol ecdysterone per g magnetic carrier from the Institute of biochemistry of the Natl.
(sample M}E) and 320 mol cholesterol per g mg Acad. Sci. of Ukraine from the plant Serratula iner- carrier (sample M}Ch).
mis L according to the procedure of Cholodova X-ray photoelectron spectra were recorded with [20]. Cholesterol was purchased from Sigma (St.
an EC-2402 XPS electron spectrometer employing Louis, MO, USA).
Mg K
exiting radiation (1253.6eV). An instrument Nanodispersed magnetite was prepared via pre- vacuum of at least 10  Torr was maintained for all cipitation from aqueous solution with KOH. The analyses. The instrument was calibrated for an surface of freshly prepared magnetite was immedi- Au4f peakbinding energy of 84.0eV. The sam- ately modi"ed by oligomerization of -aminop- ples were spread upon an aluminium template.
ropyltriethoxysilane from 5% aqueous solution XPS spectra were referenced to the maximum of the at 903C. Electron micrographs of nanodispersed C 1s spectra at 285.0 eV with additional control ofthe line position on the maximum of Fe 2p line.
Rat erythrocytes were isolated based on di!eren- tial centrifugation technique [23].
To study ecdysterone or cholesterol}magnetite conjugate binding, 2;10 cells per ml incubationmedium were stored for 15 min at 373C in thepresence of free ecdysterone or cholesterol in solu-tion. The ecdysterone and cholesterol concentra-tion was varied from 10  to 10  M. A15 min incubation with 8;10 particles of ecdys-terone or cholesterol }magnetite conjugate per cellensured then complete saturation of the bindingsites (Fig. 4a,b). The quantity of cells bindingmagnetic carrier particles was de"ned from the Fig. 3. Electron micrograph of nanodispersed magnetite with decrease in cell concentration in the supernatant after magnetically separating the cells. Optical

O.M. Mykhaylyk et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 226} 234 ing of ligands with the receptors based on the plotsregistered in the case where the concentration offree ecdysterone or cholesterol varied and the con-jugate concentration was in excess.
Concentration and time dependencies for the parameters characteristic for the signal systems ac-tivity (cAMP-dependent signal transduction andCa>-phospholipid-dependent transduction whichis associated with guanylate cyclase activity) wereregistered. 1;10 cells per ml of medium contain-ing 0.15 M NaCl in 0.01 M phosphate bu!er pH7.2, were incubated with 10 }10  M free ecdys-terone (2.5;10 }5;10  free cholesterol) orwith ecdysterone (cholesterol) immobilized on themagnetic carrier (4.8;10}2.6;10 nanodispersedmagnetite particles per cell). After 15 s incubationat 373C the cell suspension was introduced intoHClO solution, content of cGMP and cAMP ex- tracted into ethanol was analyzed using a radioim-mune analysis test system (&Amersham', UK) anda beta counter (&Beckman') [25]. Data on relativeconcentrations of cyclic purine nucleotides cGMPand cAMP are represented in coordinates sym-metric relative to the unit, namely, the positive>-axis corresponds to C/C values if C/C'1 and the negative >-axis corresponds to !C/C if C/C(1. The reference concentrations C (nmol per mg protein) in the erythrocytes suspension werefound to be 2.13 and 1.52 for cGMP and cAMP,respectively. The protein content was determined according to Bradford [26] using BSA as a stan- (N"1.95;10 cells per ml) binding ecdysterone}magnetite conjugate (a) after 15 min incubation at 373C versus concentra-tion of conjugate, (b) versus incubation time at conjugateconcentration 8;10 particles per cell, and (c) binding nanodis-persed magnetite M and ecdysterone}magnetite conjugate M-E 3. Results and discussion after 15 min incubation with magnetic carrier (8;10 particlesper cell) and with previous 30 min incubation of cells at 373C in Ecdysterone or cholesterol were covalently the presence of 0.5 nM (E#M}E) and 1 nM (2E#M}E) freeecdysterone in the incubation medium.
bound (chemisorped) on the surface of magnetiteactivated by treatment with 1,6-hexamethylen-diisocyanate. The conjugates were stable when kept density calibration curves were measured at 533 nm for a year at 43C in 0.01 M phosphate bu!er, pH and cell concentrations plotted for erythrocytes 7.2, free ecdysterone or cholesterol were not detec- and used further for the fast determination of the ted in supernatant according to spectrophotometry quantity of cells combined with immobilized prep- data. X-ray photoelectron spectroscopy (XPS) arations. The maximum fraction of these cells in the results on relative surface elemental composition absence of free ecdysterone in the incubation me- show that activation of the nanodispersed mag- dium was found to be about 80%. The method of netite sample stabilized by oligomerization of Frigyet [24] was used to evaluate the a -aminopropyltriethoxysilane (sample a) via an O.M. Mykhaylyk et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 226} 234 isocyanate procedure lead to the increase of the modi"cation from sample a to sample c in agree- surface organic layer depth. This follows from the ment with the increase in the modifying layer depth.
increase in the integral intensity of the C 1s and The increase in the absolute intensity of the C1s N 1s spectra as well as on the increase in the C/Fe components at 286.0 eV can be assigned to the ratio in sample b (Table 1). The integral intensity carbon atoms adjacent to nitrogen in the ure- ratio of Fe 2p and Fe 3p components in the XPS thane or isocyanate groups, }OC(O)NH}CH-or spectra increases whereas the intensity of the O1s }CH}N"C"O, and the increase in the abso- component at 529.6 eV from FeO decreases with lute intensity of the component at 289 eV to carbon Table 1XPS results on the relative surface elemental composition (%) and core level intensity ratios for a nanodispersed magnetite sample(a) stabilized by oligomerization of -aminopropyltriethoxysilane and sequentially modi"ed by (b) activation with 1,6-hexamethylen-diisocyanate and (c) chemisorption of ecdysterone Table 2XPS spectral features for the 3 nanodispersed magnetite samples in Table 1 C 1s spectra FWHM"0.8274 O 1s spectra FWHM"1.07 N 1s spectra FWHM"0.8274 E"peakenergy; FWHM"full-width at half-maximum height (eV); I " relative intensity (total"1.0).
O.M. Mykhaylyk et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 226} 234 involved in the urethane groups,}C(O)}NH} [27] free cholesterol in the case of cholesterol conjugate, (Tables 1 and 2). The N1 s spectra show the increase respectively) into the incubation medium inhibited in absolute and relative intensities of the compo- binding, showing evidence of speci"c binding. Ec- nents at 399.9 and 399.2 eV which can be assigned dysterone binding sites can be represented by four to the urethane bond nitrogen and to the nitrogen binding sites with equilibrium dissociation con- in the isocyanate groups, respectively [28,29], con- stant values estimated according to Frigyet method the activation procedure. Subsequent to be of 5.2;10 , 1.6;10 , 2.1;10  and chemisorption of ecdysterone (sample c) results in 0.24 nM, respectively. Competition studies in- an increase in the C/Fe ratio, decrease in the N/C dicated also that there are receptors speci"c for ratio and also in an increase in the relative intensity cholesterol on the plasma membrane of rat eryth- of the component at 399.9 eV con"rming the rocytes (Fig. 5b). The analysis revealed high-a$nity urethane bond formation.
binding components for cholesterol with K in the Figs. 4c and 5a show the results obtained on the range of 1.9;10 , 7.2;10  and 0.2 nM. The concurrent binding of immobilized ecdysterone results thus are indicative of the existence of a set of preparation with rat erythrocytes in vitro in the high-a$nity binding sites for ecdysterone and cho- range of 10 }10  M free ecdysterone. Fig. 5b lesterol on the surface of rat erythrocytes in to be represents similar results obtained for immobilized 10 }10  M concentration range.
cholesterol preparation. Incubation with ecdys- We were interested in the question whether these terone (cholesterol) immobilized on nanodispersed binding sites perform receptor function and modu- magnetite resulted in the conjugate binding by late the level of cAMP and cGMP. These second erythrocytes. Introduction of free ecdysterone (and messengers characterize cAMP- and cGMP-depen-dent signal transduction associated with adenylatecyclase and guanylate cyclase activity in the cells.
For this purpose, we compared e!ects of rapid cellactivation (within 30 min) using free ecdysteronein solution (E) or ecdysterone immobilized on mag-netic carriers (M}E). We observed immediate(within 30 s) e!ects of free ecdysterone and immobi-lized preparations on the formation of second mes-sengers cGMP and cAMP resulting in an increasein cGMP content and in a decrease in cAMP levelin erythrocytes and hence an overall signi"cant Fig. 5. Fraction of intact rat erythrocytes N/N(0) (䊏) bindingnanodispersed magnetite with immobilized (a) ecdysterone or (b) Fig. 6. Relationship between cGMP and cAMP concentrations cholesterol and the ratio of cGMP and cAMP concentrations versus time of rat erythrocytes suspension (1;10 cells per ml) (*, a) as a function of logarithmic concentration of free ecdys- incubation with 2;10  M free ecdysterone (E) or 8;10 par- terone lg([E](M)) in the incubation medium.
ticles per cell ecdysterone}magnetite conjugate (M}E).
O.M. Mykhaylyk et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 226} 234 increase in the relationship between these purine These results can be explained in terms of rapid nucleotide concentrations (Fig. 6). An important (within 15 s) increase in guanylate cyclase activity feature revealed is the termination of the cell activa- and decrease in adenylate cyclase activity in rat tion after 30 min incubation. At concentrations of erythrocytes stimulating the cGMP and declining 2;10  free ecdysterone and 8;10 conjugate cAMP synthesis in response to binding of ecdys- particles per cell a maximal increase of the basal terone and cholesterol (Fig. 7a and b). Similar ef- [cGMP]/[cAMP] relationship reached 800% and fects were observed in the concentration ranges 400% for free ecdysterone and magnetite}ecdys- of 5;10 }5;10  and 5;10 }1;10  M terone conjugate, respectively.
free ecdysterone corresponding to speci"c binding Fig. 7. Relative concentrations C/C (or!C/C if C/C(1) (a}e) of cyclic purine nucleotides cGMP (䊏) and cAMP (䉭) and (a
}e
) their ratio [cGMP]/[cAMP] as a function of logarithmic concentrations of (a, a
) free ecdysterone E (b, b
) free cholesterol Ch, (c, c
)conjugates of ecdysterone M}E, (d, d
) cholesterol M}Ch and (e, e
) nanodispered magnetite M 15 s after their inroduction intosuspension of rat erythrocytes (10 cells/ml). Reference concentrations (nmol/mg protein) were [cGMP]"2.13 and [cAMP]"1.52.
O.M. Mykhaylyk et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 226} 234 with erythrocytes plasma membrane (Fig. 5a and b) A maximal increase of the basal [cGMP]/[cAMP] nanocarriers could eliminate or minimize detrimen- relationship reached 425% for 2.5 M free choles- tal side e!ects (in particular, carcinogenic) when terol (Fig. 7b, b
).
high concentrations of these compounds are used.
The concentrations of tested second messengers Magnetic conjugates of C-steroid may have util- change in a similar manner in response to the ity in the treatment of a variety of malignancies. It introduction of free and immobilized ecdysterone was shown that the active metabolite of vitamin D, (or cholesterol) preparation into erythrocyte sus- 1,25-dihydroxyvitamin D3 (calcitriol), which has pensions; namely, rapid increase in cGMP concen- a structure similar to that of ecdysterone and cho- tration and decrease in cAMP concentration occur lesterol inhibits the growth of prostate cancer and (Fig. 7c, c
, d and d
). Similar change of tested para- breast tumor cell lines [34]. Considerable workhas meters in response to the free and immobilized been done to develop vitamin D analogs to maxi- ecdysterone and cholesterol preparations is an mize anticancer and to reduce calcemic activity evidence of high speci"c activity of immobilized [35,36]. Further research will have to evaluate the preparations. There were no extremes observed in clinical signi"cance of the C-steroid rapid mem- the changes of [cGMP]/[cAMP] ratio when the brane e!ects.
reference magnetite sample with chemisorbedglycine was used (Figs. 7e and e
).
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