New Findings Concerning the Mutual Action of Hormones and Receptors

Results and Discussion

Experiments showed that ME as well as tyrosine (TYR) (as a main component of ME) emit electrons (e_aq⁻) from their singlet excited state by UV-irradiation in aqueous solution (pH~7.4; 37°C). The observed quantum yields, Q (e_aq⁻), decrease with increasing substrate concentration (Table I). This unusual effect was attributed to the fact that both compounds form associates (unstable complexes of molecules). These complexes were spectrophotometrically established at concentrations >10⁻⁷ mol/l. The associates consist of molecules prevailing in the ground state, which react with a proportion of the ejected e_aq⁻. The reaction rate constants are: k(e_aq⁻ + TYR)=3.4×10⁸ l mol-1 s⁻¹ (11) and k(e_aq⁻ +ME) ≈ 5.3×10⁹ l mol⁻¹ s⁻¹ (calculated on the basis of the amino acids sequence); and for 17βE₂ k (e_aq⁻ + 17βE₂) ≈ 2.7×10¹⁰ l mol⁻¹ s⁻¹ (11).

It has been recently established that hormone transients resulting by e_aq⁻ emission and being in a nascent state can be regenerated by electron transfer from an efficient electron donor, such as ascorbate (7, 8). Based on the outcome of these studies, the interaction of ME with vitamin C in air-free aqueous solution (pH~7.4; 37°C) was investigated. The process was followed by HPLC analysis, where the remaining part (Rm, %) of the particular substrate, individually and in a mixture of both, ME and vitamin C, before and after each UV-treatment was registered as a function of absorbed UV dose (hη/l). The obtained results are presented in Figure 1.

From the course of the ME photolysis, registered by an HPLC peak at 10.3 min (retention) (curve A, Figure 1) and this of the mix of ME and vitamin C, registered also by the ME peak (curve A1, Figure 1), it is obvious, that ME is partly regenerated by electron transfer from vitamin C to ME transients. Curve B, Figure 1 (HPLC peak at 4.6 min) shows the very strong vitamin C degradation as a function of absorbed UV dose. However, curve B1, Figure 1, expressing the HPLC-peak of vitamin C in the presence of ME, shows at the same UV dose range, even a much higher maximum. Very likely the transients of ME and vitamin C have the inclination to form complexes, which facilitate the electron transfer process within both substrates. By extrapolation of curve A1 to nilUV dose and considering the calculated Qi (Rm) values, a ME regeneration of 14.6% was found (see insert I, Figure 1).

Similar experiments were also performed using 5×10⁻⁵ mol/l vitamin C individually and in mix with 5×10⁻⁵ mol/l ME (insert II, Figure 1). It is interesting to note that the corresponding curve A1 did not show any maximum. The maximum of curve B1 (expressing vitamin C in mix with ME as a function of dose), was much smaller compared to that shown in Figure 1. This effect might be a sequence of the higher vitamin C concentration used and that of the reaction rate constants of the involved components mentioned above. The obtained initial quantum yields Q(Rm), and the regeneration of ME of 52.8% are given in insert II, Figure 1.

As well-known the steroid hormones are activated by complexing with the corresponding receptor. For the 17βE₂ action the corresponding receptors, ERα and ERβ are responsible. Both receptors are complexing to 17βE₂ with similar activity. It was now of interest to get a deeper insight into the very complicated mechanisms of the hormone receptor action and, in addition, to contribute, if possible, to a better understanding of the observed fast communication pathway of hormones on the basis of e_aq⁻ transfer process (4).

In order to get information about the interaction of ME with 17βE₂, two series of experiments were performed. In I series 2.5×10⁻⁵ mol/l of each substrate were used, which were UV irradiated individually as well as in mixture of both. In II series the substrates concentration was two-fold each. Based on HPLC analysis Figure 2 shows the reminder (Rm, %) of the I series of UV irradiated ME and 17βE₂ samples in air-free polar media (water/ethanol 40/60 v/v) at pH~7.4 and 37°C, as a function of absorbed UV dose. The course of the curves representing the remainder (Rm, %) separately of each substrate (curves A and B) as well as of the given mixture (curves A1 and B1) are shown in Figure 2. It is obvious that the Rm-value of ME strongly decreases with absorbed UV-dose, comparing curve A and A1. This illustrates that ME is acting as an e_aq⁻ donor for 17βE₂. Simultaneously, the Rm-value of 17βE₂ is increasing in the presence of ME, comparing curve B with B1, which demonstrates, that 17βE₂ is partly regenerated by e_aq⁻ transfer from ME. Thereby, 7.5% ME is consumed for the regeneration of 2% 17βE₂.

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Figure 1.

HPLC analysis: Substrate remainder (Rm, %) as a function of absorbed UV-dose (hη/l) of (A) 5×10⁻⁵ mol/l ME; (A1) 5×10⁻⁵ mol/l ME in mix with 2.5×10⁻⁵ mol/l vitC; (B) 2.5×10⁻⁵ mol/l vitC; (B1) 2.5×10⁻⁵ mol/l vitC in mix with 5×10⁻⁵ mol/l ME in aqueous solution (pH~7.5) at 37°C. Insert (I). [Qi(Rm)] of the above systems. Insert (II). Similar data of studies using 5×10⁻⁵ mol/l ME and 5×10⁻⁵ mol/l vitC.

From the course of the respective curves resulting of II series (not graphically presented) it could be determined that 1% ME was consumed as electron donor for regeneration of 2.3% 17βE₂ (see insert II, Figure 2) The obtained results demonstrate that several reaction mechanisms are involved in the interaction of ME (used as a receptor model) and 17βE₂ (hormone representative). Thereby their reactivity with e_aq⁻ was already mentioned.

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Figure 2.

HPLC analysis. Substrate remainder (Rm, %) of substrates dissolved in water/ethanol (40/60 v/v), airfree; pH~7.5. Series I. (A) 2.5×10⁻⁵mol/l ME, (A1) 2.5×10⁻⁵ mol/l ME + 2.5×10⁻⁵ mol/l 17βE₂; (B) 2.5×10⁻⁵ mol/l 17βE₂ (B1) 2.5×10⁻⁵ mol/l 17βE₂ + 2.5×10⁻⁵ mol/l ME. Series II. The same kind of studies, but 5×10⁻⁵ mol/l of each substance was applied. The obtained data of both series are given as insert.