Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes.

The presence of a divalent cation is essential for the specific binding of [3H]oxytocin to particulate receptor preparations of the mammary gland of the lactating rat. Oxytocin binding was potentiated in increasing order by divalent zinc, magnesium, nickel, manganese, and cobalt, but was negligible in the presence of divalent calcium, copper, and iron. The apparent K, values for oxytocin-receptor interaction in the presence of optimal concentrations of metal ions ranged from 3.1 to 8.6 X 10’ M-~. The metal ions did not affect site-site interactions among oxytocin receptors, as evidenced by linear Scatchard plots, Hill coefficients of 1, and the lack of effect of unbound oxytocin on the dissociation rate constant of the oxytocin-receptor complex. The kinetics of the association and dissociation of the hormone *receptor complex showed a fast step for the binding of metal ion followed by a slow rate-determining step for the binding of oxytocin. The active divalent metal ions appear to affect oxytocin binding by two separate processes. 1) Increasing concentrations of divalent nickel, magnesium, and manganese caused an increase in the concentration of binding sites available for oxytocin, while the aftinity for the hormone was not changed. 2) Increasing amounts of cobalt increased the affinity of the receptor site for oxytocin, but did not affect the concentration of sites available for oxytocin binding. Because combinations of maximal concentrations of both types of metal ions were not additive with respect to the concentration and affinity of oxytocin-binding sites, metal ions appear to bind to identical sites. We postulate that the oxytocin receptor possesses two distinct regions for metal ion interaction. The binding of metal ion to Region A (availability) results in a receptor with the maximum number of available binding sites, but with a low affinity for oxytocin. The binding of metal ion to Region B (binding) results in a receptor site of low availability but of potentially high affinity for oxytocin. Binding of metal ions to both sites results in a receptor of high affinity and full availability. This proposed model is consistent with the results of studies of neurohypophysial hormone action on isolated target tissues.

, and isolated mammary strips (7,&J). On the other hand, Mg2+ does not potentiate the vasoconstrictor actions of serotonin, angiotensin, or epinephrine (8). Thus, the actions of Mg2+ on smooth muscle appear to be unique for neurohypophysial peptides.
The binding of ["Hloxytocin to mammary particulate fractions reflects oxytocin-receptor interaction (10)(11)(12). This binding requires the presence of a divalent cation other than Ca2+; Soloff and Swartz (10)  These measurements are based on the assumption that the binding sites on the oxytocin molecule for Cu*' are similar to those for the other divalent metal ions and the values obtained are considered approximations of the true binding constants.

Specific
Binding of Oxytocin with Different Metal Ions-As shown in previous studies with particulate fractions (10) and isolated cells (18) from the mammary gland of the lactating rat, the interaction of oxytocin and receptor sites results in linear Scatchard plots. The binding of oxytocin to mammary receptor sites in the presence of increasing concentrations of metal ions has been examined with Co2+, Mn2+, Ni", Mg'+, Zn2', Ca'+, and CL?+ and analyzed according to Scatchard (19). Oxytocin binding was indistinguishable from nonspecific binding in the presence of Zn2+ (0 to 10 m&f), Ca2+ (0 to 5 mM), and Cu2+ (0 to 5 mM), but was potentiated by Co*+, Mn*', Ni*+, and Mg2+ (Fig. 1). The binding affinity for oxytocin was maximal with 5 mM Co2+, apparent K, = 8.6 X 10' M-l. The apparent K, values with 5 mM Mn'+, Mg'+, and Ni2+ were 4.7, 4.3, and 3.1 X lOa M-l, respectively.
The amount of oxytocin bound to 6 mg of particulate protein/ml was near maximal with concentrations of Mn" near 5 mu; the value of R,,, with 5 mM Mn*+ was 0.85 nM compared with the theoretical maximum of 1.05 nM shown in the inset to Fig. 1. A decrease in the concentration of either Mn*', Mg'+, or Nil+ resulted in a reduction in the maximum number of available binding sites for oxytocin; the apparent K, values, as indicated by the slopes of the Scatchard plots, were not different within experimental error ( Fig. 1). In contrast, with 0.5 to 5 mM Co2+, the maximum number of binding sites available for oxytocin remained constant, but the affinity of the receptor sites for the hormone decreased as the [Co"'] was reduced ( Fig. 1). Scatchard plots obtained with [3H]oxytocin alone were identical with those determined with combinations of labeled and unlabeled oxytocin. Therefore, radiolabeled oxytocin was indistinguishable from unlabeled hormone. To determine whether metal ions affect site-site interactions among the oxytocin-binding sites, the equilibrium binding data were plotted according to the Hill transformation  w" E 0 2 5 2 0.6 bound with a given and saturating concentration of hormone, respectively, gave Hill coefficients that were close to 1 (0.89 to 1.07) with correlation coeffkients greater than 0.98 (Fig, 2). Parallel lines were obtained with different concentrations of Co", whereas a single line was obtained with increasing concentrations of Mn2+ and Mp (Fig. 2). The linear Scatchard plots and Hill coefficients of 1 indicate that oxytocin is bound to a single class of binding sites that show no significant cooperativity in the presence of metal ions.
Dissociation Rate Studies with Mn2+-The dissociation of the oxytocin-mammary receptor complex was studied by diluting an equilibrium mixture of the bound hormone either with buffer alone or with buffer containing nonradioactive hormone.
Under these conditions, reassociation of the hormone -receptor complex was negligible and the data could be analyzed by assuming a unimolecular dissociation where R + Oxy $R.Oxy (1) For this mechanism k, = In 2/t1/2 (2) where tl/2 is the half-time for the dissociation determined by plotting the log of the percentage of dissociation uersus time. The half-time for a series of dissociation experiments was 15.2 f 3.1 min with initial oxytocin concentration between 5 and 50 nM (Table I). In one experiment, the initial receptor concentration was halved with no significant change in tl12 (Table   I). No significant differences in dissociation rates were found with or without a lOO-fold excess of nonradioactive oxytocin in the diluent buffer (Table I).
Because the dissociation rates were not accelerated by the presence of an excess of oxytocin, retention of free-hormone in an insoluble compartment is not a rate-determining feature of this system (21). Thus, we can eliminate any effect on the kinetics due to ligand distribution between the soluble and insoluble phases.
Association Rate Studies with Mn2'-The specific binding the concentration of receptor (Fig. 3A) and of oxytocin (Fig.  3B). This suggests that the binding of oxytocin to the particulate receptor is a second order process. The mechanism which fits the data is shown below: .+0x+ R.Oxy (3) ,where R is the receptor site, Oxy is oxytocin, and R. Oxy is the complex formed. Determinations of kf were made at early times (0 to 5 min) to minimize contributions from the dissociation reaction. The integrated equation for this mechanism, including a term for the dissociation reaction, may be expressed as where q = @da, /I = -(k, + R" + Oxy'), and a = (R') . (Oxy') and where the superscript zero indicates initial concentrations and the subscript t indicates the concentration at the time of the measurement.
The relationship between the lefthand term of the integrated rate equation, F(c), and t was linear during the first 5 min of association. Association rates also were linear during the fast 5 min with the concentrations of Co'+, N?', and Mg2+ shown in Fig. 1. Analysis of the data by assuming a two-step mechanism by the method of Strickland et al. (22) gave rate constants that were internally inconsistent. The average forward rate constant with 5 mM Mn2+ for five experiments in which the initial oxytocin concentration was varied was 3.7 f 0.3 (S.E.) x lo5 M-' s-' (Table II). Ions-We have examined the kinetics of association and dissociation of oxytocin in the presence of increasing amounts of Co2+, Mg"+, and Ni" as well as Mn2+ (Table III). The rates of association and dissociation of oxytocin were unchanged by increasing concentrations of Mn2+, Mg2+, and Ni2' (Table III). These findings are consistent with the results from studies of the binding under steady state conditions; namely, these metal ions caused an increase in the concentration of receptor sites but did not affect the magnitude of the equilibrium association constant. In contrast, increasing concentrations of Co2+, caused a small increase in the association rate constant and a decrease in the dissociation rate constant. These effects of increasing cobalt concentration on the rate constants may account, in part, for the increase in stability of the oxytocin.receptor complex in the presence of increasing amounts of cobalt.

Delayed Additions of Metal
Ion-The binding of ["Hloxytocin to a mammary receptor preparation was negligible for up to 30 min in the absence of metal ion (Fig. 4). The addition of Mn'+, to a final concentration of 5 mM, resulted in rapid interaction of oxytocin and receptor with an association rate constant identical with that obtained when [3H]oxytocin and Mn"+ were added simultaneously (Fig. 4).  buffer containing 5 mu of the appropriate metal ion resulted in no experimentally detectable dissociation of oxytocin by 1 min; however, dilution with buffer containing no metal ion or containing no metal ion and 1 mM EDTA resulted in the dissociation by 1 min of 43 and 62 to 72%, respectively, of the oxytocin bound (Fig. 5).

Effect of Metal
Metal Ion Binding to Oxytocin-To assess the affinity of oxytocin for metal ions, we determined the overall binding constants of a series of divalent metal ions. The results of pH titration studies indicated that the affinity of most of the Mg" 3 x lo3 Ca" 2 x lo3 Ni" 2 x lo3 0' Upper panel: 5 mM Co'+, 0, 5 mM Co'+ and 5 mM Mn*+, 0, 5 mM Co'+ and 5 mM Ni*+, A; and 5 mM Co'+ and 5 mM Mg'+, l . Lower panel: 5 mM Mn'+, 0; 5 mM Mn'+ and 5 mM CO", 0, 5 mM Mn*+ and 5 mM Nil', A; and 5 mhi Mn*+ and 5 mM Mg'+, 0. divalent metal ions for oxytocin was too low to measure with reasonable amounts of hormone by this technique.
Copper binds to both oxytocin and vasopressin, with a binding constant of about 5 X lo5 M-' at pH 7 (23). We used a solid state Cu2+ electrode to measure the binding of Cu2+ to oxytocin. We then added another divalent metal ion and determined the relative binding constant for the metal ion based on its ability to displace Cu2+ from oxytocin (Table IV). Although these values are only approximate, they indicate clearly the relatively low affinity of oxytocin for most divalent metal ions.
Association of Oxytocin to Mammary Membrane Particles with Combinations of Divalent Metal Zons-To determine whether the different metal ions bind to the same site when enhancing oxytocin binding, we examined combinations of maximal concentrations of metal ions with 5 mM Mn'+ and 5 mM Co*+. The results are plotted as l/bound oxytocin (l/B) uersus l/free oxytocin (l/F) for either Co'+ or Mn"+ with MnZt or Co'+, Ni2+, and Mg"+ as the second metal ion (Fig. 6). Specific binding was not significantly increased by maximal combinations of metal ions when compared with either Mn2+ or Co2' alone (Fig. 6).
Effect of Ca'+ on the Binding of Oxytocin in the Presence of 5 mM Mg'+-Calcium is required for the contractile response of mammary strips to oxytocin (8, 24,25). Because the binding of oxytocin to mammary receptor sites was not affected by Ca"+ ( Fig. 7, inset), Ca2+ appears to be involved in molecular events distal to the activation of the receptor. However, the binding of [3H]oxytocin to mammary receptor sites with 5 mM Mg2+ was inhibited by 0.2 to 1.0 mM Ca" (Fig. 7). Concentrations of Ca*' greater than 1 mu were not inhibitory.
Scatchard plots of oxytocin binding with 5 mM Mg*+ and increasing concentrations of Ca2+ indicate that the inhibitory activity of 0.2 to 0.6 mM Ca2+ was due to a reduction in the concentration of binding sites available for oxytocin (Fig. 8). The affinity of the receptor sites for oxytocin was not changed, as shown by the parallel Scatchard plots for 0, 0.2 and 0.4, and 0.6 IDM Ca2+. In agreement with these findings, association and dissociation rates of the hormone-receptor complex in the presence of 5 mM Mg2+ were not affecte.d by Ca2+ concentrations of 0.5 and 0.6 mM. Ca*+ (1 mM) caused an increase in the affinity of the receptor sites for oxytocin as well as a further decrease in the maximal concentration of receptor sites (Fig. 8). This increased affinity for oxytocin may explain why concentrations of Ca*' greater than 1 mM were not inhibitory with the concentrations of oxytocin and receptor used (Fig. 7).
All of the Scatchard plots were linear (Fig. 8), which indicates that oxytocin was bound to a single class of independent sites at the concentrations of Ca" tested. The observation that calcium can affect both the concentration as well as the affinity of binding sites for oxytocin is consistent with the effects of other metal ions that we have examined.

DISCUSSION
In the absence of active divalent metal ions, specific oxy-o.02d 0 2 3 4 5 Co (mM) tocin binding to mammary particulate material was only slightly greater than nonspecific binding levels. For example, in the absence of metal ion, the bound/free ratios of oxytocin never exceeded 0.05 when 0.01 to 0.1 no oxytocin was bound (compare with data in Fig. 1). Thus, the presence of an appropriate divalent metal ion is an absolute requirement for significant oxytocin binding.
The effect of the metal ion was rapid compared with oxytocin binding. Within 10 s of the addition of Mn*+ to a preequilibrated mixture of receptor and oxytocin, hormone binding occurred at a rate identical with that obtained when oxytocin, metal ion, and receptor were incubated simultaneously (Fig. 4). Divalent metal ion complexation is known to occur rapidly with typical association rate constants of 2 x lo6 to 3 X lo7 M-l SK' (26). Metal ion was required to prevent dissociation of the hormone. receptor complex because dissociation of the oxytocin. receptor complex was greatly accelerated when metal ion was complexed with EDTA (Fig. 5).
In spite of the distinct differences between the effects of Co*+ and the other divalent cations, all of the divalent metal ions probably bind to the same sites because combinations of maximal amounts of Co*+ and other metal ions were not additive with respect to the concentration and affinity of oxytocin-binding sites (Fig. 6). Calcium alone did not affect oxytocin binding, but, in combination with 5 mM Mg2+, 0.2 to 0.6 mM Ca2+ caused a reduction in the concentration of oxytocin receptor sites (Fig. 8). Higher concentrations of Ca2+ in the presence of 5 mu M$+ increased the affinity of the receptor for oxytocin (Fig. 8). These findings indicate that, under certain conditions, a single metal ion, Ca*+, can affect both the concentration and the affinity of receptor sites for oxytocin. Therefore, any model describing the effects of metal ion must be consistent with this dual action of Ca'+.
Although metal ions may potentiate biological effects by modifying the conformation of the hormone directly, this is unlikely to be the case for the oxytocin-mammary receptor system. Neurohypophysial hormones form strong complexes with Cu*+ (23), yet copper is inactive in the oxytocin-mammary receptor system. Although those divalent metal ions that potentiate oxytocin-receptor interaction have a lower affinity than Cu2+ for oxytocin (Table IV), we estimate that, under most experimental conditions, about 95% of the oxytocm would be complexed with metal ion. It is unlikely, however, that the initial effect of the metal ion is upon the conformation of oxytocin. If metal ion binding to oxytocin were of sign& cance, then Kl2 M+Oxy = MO,,,;5 M.0xy.R where M is the metal ion, Oxy is oxytocin, R is the receptor site, Me Oxy is the metal ion. oxytocin complex, and Mm Oxy . R is the complex of all three components, the equal sign indicates a fast pre-equilibrium (1 to 2) and the arrows indicate the rate-determining step (2 to 3). If the assumption is made that Me Oxy is present as a steady state intermediate and that the concentration of free receptor is in the nanomolar range, the observed forward and reverse rate constants may be expressed as k, = I&& [M] and K, = k~2 (5) where K12 is the association constant for oxytocin-metal ion binding. Were Mechanism 4 correct, then the observed forward rate constant should be directly dependent on the concentration of free metal ion. However, there was no increase in the observed association rate constant with increasing Mn2+ and Mg"+ concentrations (Table III). With Co'+, there was only a 26% increase in the observed forward rate constant with a &fold increase (1.0 to 5.0 mM) in cobalt concentration (Table III). Furthermore, Scatchard analysis of oxytocin binding in the presence of cobalt showed the same concentration of receptor available to bind oxytocin at all cobalt concentrations. The fact that cobalt concentration was not limiting in this regard eliminates Mechanism 4 as a realistic model. We conclude that metal ions bind to the receptor site first and then a complex is formed between the receptor site and the hormone. Our data cannot discriminate between a mechanism involving metal ion interaction with the receptor site only or metal ion interaction with both the receptor site and oxytocin. The Scatchard plots as well as the kinetic data are strong indications that receptor-metal ion interactions must occur prior to hormone binding.
We have developed a model that is consistent with our data in which the active metal ions affect the conformation of the receptor site for oxytocin (Fig. 9). The receptor complex is postulated to consist of at least two subunits or domains and to contain at least two distinct metal ion binding regions: A, for availability and B, for binding ( Fig. 9, I). The A site is part of an "inhibitory" subunit that blocks the access of oxytocin to the receptor binding site. When metal ions bind to the inhibitory subunit at Site A, the inhibitory subunit dissociates from the binding subunit and exposes the binding site for oxytocin ( Fig. 9, II). Binding of metal ion to the second binding region, B, results in a conformational change at the can be bound to species II with low affinity and species IV with high affinity.
The meaning of the symbols is described in the text and summarized in Table V. hormone binding site (Fig. 9, III) and allows for high affinity oxytocin binding when the inhibitory subunit is dissociated (Fig. 9, IV). For the sake of simplicity, we have postulated that the inhibitory portion of the receptor complex is a distinct subunit. It is possible that both the A and B sites are on the same molecule.
The active metal ions can bind to both A and B regions. However, the B region has a greater affinity for Mn2+ and Me than does the A region, so that at low concentrations of these ions the B region is occupied before the A region. This results in a receptor site with a maximal affinity for oxytocin, but with limited accessibility because of the steric hindrance posed by the presence of an undissociated inhibitory subunit (Fig. 9, I + III). As the concentration of Mn2+ or Mg2+ increases, the A sites on the receptor complex progressively become occupied. This results in a greater number of dissociated inhibitory subunits and, consequently, an increase in the availability of the high affinity sites for oxytocin (Fig. 9, III --, IV). The increase in the total number of binding sites for oxytocin as the concentration of Mn"+ or Mg" is increased is proportional to the occupancy of the A region by metal ion. This is reflected in the parallel shift to the right of the Scatchard plots for oxytocin-receptor binding in the presence of increasing amounts Mn*+, Mg2+, or Ni2' (Fig. 1).
Cobalt, on the other hand, has a greater affinity for the A site, so that at low concentrations of Co*+, the inhibitory subunit is dissociated; this results in the maximum availability of binding sites, but the receptor site is in a conformation that has a relatively low affinity for oxytocin (Fig. 9, I -+ II). As the concentration of Co2+ is raised, the B site progressively becomes occupied. This results in an increasing concentration of receptors with maximal affinity for oxytocin (Fig. 9, II + IV). The increase in affinity is proportional to the relative amount of receptor subunits binding metal ion at Site B. Thus, Scatchard plots of oxytocin binding to receptor with increasing concentrations of Co'+ will show an increasing association constant for oxytocin binding with a maximal concentration of receptor sites always available for binding. Further support for our model is provided by the observation that Ca2+, while not effective by itself in potentiating the binding of oxytocin to mammary receptor sites, was able to exert separate effects on both the affinity and concentration of receptor sites. Calcium, 0.2 to 0.6 mM, reduced the concentration of accessible binding sites, presumably by competing with Mg2+ at the A metal-binding region. Concentrations of Ca2+ greater than 1 mM caused an increased affinity of the receptor site for oxytocin. This effect may be either due to the direct interaction of Ca" with the B site or to the effect of Ca2+ on the concentration of Mg2+ available for binding to the B site.
The effects of combinations of metal ions near maximal concentrations were not additive (Fig. 6). These findings add further support to the model, which proposes that the divalent metal ions bind to identical regions, A and B, when enhancing oxytocin binding.
Our model is consistent with the results of studies of neurohypophysial hormone action on isolated target tissues. These studies have suggested that Mg'+ potentiates the action of oxytocic peptides by increasing the affinity of the receptor sites for the peptide. For example, the addition of Mg"+ to the medium bathing isolated mammary strips caused a parallel displacement to the left of log dose-response curves for a series of oxytocin analogues (24). The maximum response to oxytotin was unchanged by the addition of Mg2+ (24). Comparable results have been obtained for a number of oxytocin analogues with the isolated uterus (5-7) and isolated blood vessels (8,9). Studies with other oxytocin analogues have shown that both the maximal response of the target tissue as well as the affinity for the hormone was diminished in the absence of Mg2+ (7,9). It has been assumed that this reduction in response was due to a decrease in the intrinsic activity of the peptides in the absence of Mg2+ (7,9). However, it is equally possible that the reduced response was the result of a reduction in the concentration of available receptor sites for the oxytocic peptides. Therefore, M$+ may affect both the affinity and concentration of oxytocin receptor sites in intact, isolated target organs as well as in particulate fractions from the mammary gland. Our observations on the comparative activities of Mn"+ and Mgz' are consistent with results showing that 0.1 mM Mn2+ was more effective than 0.5 mM Mg"+ in potentiating the potency of several oxytocin analogues in the isolated uterus (5). The order of effectiveness of divalent metal ions in increasing the affinity of the mammary receptor sites for oxytocin was Co"+ > Mnz+ > Ni"+ > Mgz+ > Zn2+. No specific binding occurred in the presence of Ca2+, Cu2+, and Fe'+. This order is the same as that reported by Schild (27) for the effect of metal ions on lysine vasopressin activity on the depolarized uterus of the rat.
There is no clear relationship between the relative activities of the metal ions and size as estimated by their crystal ionic radii and the strength of their coordination complexes (28). The order of magnitude of the equilibrium constants for complexation of divalent metal ions with nitrogen donor ligands (Irving-Williams series) is Ca = Mg c Mn < Co < Ni < Cu < Zn (29). Although there is some correlation between the rank order of cobalt, manganese, and magnesium and their effectiveness in promoting oxytocin-receptor binding, it is probably inappropriate to generalize from simple complexes to complicated systems. Multiple binding sites may be arranged in specific configurations which we can neither examine nor predict in the system under study. Although the present studies do not delineate the precise mechanisms by which metal ions affect the activity of oxytocin, they provide a model that serves to explain the role of metal ions in potentiating oxytocin action.