Allosteric modulation of semicarbazide-sensitive amine oxidase activities in vitro by imidazoline receptor ligands

Abstract

1. Evidence indicates that imidazoline I(2) binding sites (I(2)BSs) are present on monoamine oxidase (MAO) and on soluble (plasma) semicarbazide-sensitive amine oxidase enzymes. The binding site on MAO has been described as a modulatory site, although no effects on activity are thought to have been observed as a result of ligands binding to these sites. 2. We examined the effects in vitro of several imidazoline binding site ligands on activities of bovine plasma amine oxidase (BPAO) and porcine kidney diamine oxidase (PKDAO) in a spectrophotometric protocol. 3. While both enzymes were inhibited at high concentrations of all ligands, clonidine, cirazoline and oxymetazoline were seen, at lower concentrations, to increase activity of BPAO versus benzylamine, but not of PKDAO versus putrescine. This effect was substrate dependent, with mixed or biphasic inhibition of spermidine, methylamine, p-tyramine and beta-phenylethylamine oxidation observed at cirazoline concentrations that increased benzylamine oxidation. 4. With benzylamine as substrate, clonidine decreased K(M) (EC(50) 8.82 microm, E(max) 75.1% of control) and increased V(max) (EC(50) 164.6 microm, E(max) 154.1% of control). Cirazoline decreased V(max) (EC(50) 2.15 microm, E(max) 91.4% of control), then decreased K(M) (EC(50) 5.63 microm, E(max) 42.6% of control) and increased V(max) (EC(50) 49.0 microm, E(max) 114.4% of decreased V(max) value). 5. Data for clonidine fitted a mathematical model for two-site nonessential activation plus linear intersecting noncompetitive inhibition. Data for cirazoline were consistent with involvement of a fourth site. 6. These results reveal an ability of imidazoline ligands to modulate BPAO kinetics allosterically. The derived mechanism may have functional significance with respect to modulation of MAO by I(2)BS ligands.

Figure 1

Preliminary screening results for effects of imidazoline ligands on benzylamine oxidation by BPAO (a) or putrescine oxidation by PKDAO (b). Benzylamine (740 μM) was incubated with BPAO (1 mg ml⁻¹), or putrescine (288 μM) with PKDAO (2.5 mg ml⁻¹) in the presence of test compounds (0.1 μM–1 mM) and the absorbance at 498 nm was monitored continuously. Initial rates, obtained from a single assay at each concentration of test compound, are expressed as a percentage of those in relevant control experiments. Estimates of IC₅₀ values, obtained by nonlinear regression, for those drugs showing concentration-dependent inhibition are, versus BPAO, 2.5 μM (guanabenz), 4.8 μM (guanfacine), 41 μM (2-BFI) and 47 μM (efaroxan), and versus PKDAO, 33 μM (guanfacine), 47 μM (guanabenz), 168 μM (2-BFI), 233 μM (oxymetazoline), 576 μM (cirazoline) and 748 μM (clonidine).

Figure 2

Effects of the I₁R ligand moxonidine (MOX), the I₂BS ligand idazoxan (IDZ) and the I_2ABS ligand amiloride (AM) on the ability of cirazoline (CIR; 60 μM) to potentiate oxidation of benzylamine (300 μM) by BPAO. Moxonidine, idazoxan or amiloride was added to assay wells containing BPAO (0.167 mg ml⁻¹) and either water (−CIR) or cirazoline (+CIR). Plates were warmed to 37°C and reactions were started by the addition of benzylamine. Initial rates of change in absorbance at 498 nm are expressed as a percentage of that in a control experiment containing only BPAO and benzylamine. Data are the mean±s.e. mean values from triplicate determinations; error bars do not exceed symbol size.

Figure 3

Effects of cirazoline (0.1 μM–3 mM) on oxidation by BPAO of benzylamine (BZ; 300 μM), p-tyramine (TYR; 135 μM), β-phenylethylamine (PEA; 275 μM), methylamine (MA; 420 μM) or spermidine (SPD; 50 μM), concentrations approximating 50% of their respective K_M values. Amines were incubated with BPAO (0.33 mg ml⁻¹) and water (controls) or cirazoline, and absorbance was monitored continuously at 498 nm. Data are mean±s.e. mean initial rates from duplicate determinations and are expressed as percentages of relevant control values, determined in the absence of cirazoline. EC₅₀ and IC₅₀ values were obtained by nonlinear regression analyses of either partial data sets (0.1–100 μM, inclusive, and 100 μM–3 mM, inclusive) for benzylamine, or of the entire data sets for other substrates. Uniphasic sigmoidal curves were fitted to data for benzylamine and spermidine with a standard four-parameter logistic equation, while biphasic sigmoidal curves were fitted to data sets for other substrates with an equation describing two-site competition (GraphPad Prism 4). EC₅₀ and IC₅₀ values thus determined were as follows: BZ, 9.1 μM (activation) and 3.5 mM (inhibition); SPD, 19.1 μM; MA, 8.7 μM and 7.7 mM; TYR, 22.4 μM and 2.9 mM; PEA, 35.4 μM and 4.8 mM. For those substrates for which biphasic inhibition was apparent, the fraction of total amine turnover inhibited by the ‘high-affinity' component of cirazoline's effect was 14% (MA), 60% (TYR) and 36% (PEA).

Figure 4

Effects of cirazoline on the kinetic constants K_M (a) and V_max (b) for benzylamine turnover by BPAO. Benzylamine (10 μM–3 mM) was incubated at 37°C with BPAO (0.167 mg ml⁻¹) in the presence of water (controls) or cirazoline (1 μM–10 mM) and absorbance at 498 nm was monitored continuously. Initial rates were plotted versus substrate concentrations and kinetic constants were obtained following fitting of rectangular hyperbolae by nonlinear regression to mean±s.e. mean velocity data from triplicate measurements. Data, which are from one experiment representative of three similar experiments, thus show the best-fit±s.e. kinetic constants, where standard errors are asymptotic standard errors of best-fit values. EC₅₀ values in this representative experiment, obtained following nonlinear regression of the kinetic constant replots, are 5.9 μM (effect on K_M; 95% confidence intervals, 3.0–11.6 μM) and 44.0 μM (effect on V_max; 95% confidence intervals, 39.6–48.8 μM). Maximal effects are exhibited as a 55.2% reduction in K_M and a 10.0% increase in V_max.

Figure 5

Effects of clonidine on the kinetic constants K_M (a) and V_max (b) for benzylamine turnover by BPAO. Benzylamine (10 μM–3 mM) was incubated at 37°C with BPAO (0.167 mg ml⁻¹) in the presence of water (controls) or clonidine (1 μM–50 mM) and absorbance at 498 nm was monitored continuously. Analyses were completed as described in the legend to Figure 4. EC₅₀ values in this representative experiment, obtained following nonlinear regression of the kinetic constant replots, are 7.0 μM (effect on K_M; 95% confidence intervals, 3.3–14.6 μM) and 134 μM (effect on V_max; 95% confidence intervals, 90.1–198 μM). Maximal effects are exhibited as a 21.8% reduction in K_M and a 56.2% increase in V_max.

Figure 6

Replots of kinetic constants for oxidation of benzylamine by BPAO in the presence of high (inhibitory) concentrations of cirazoline or clonidine. K_M and V_max values for benzylamine oxidation were obtained by nonlinear regression analysis of velocity data, as described in the legend to Figure 4. Constants determined for assays carried out in the presence of cirazoline (300 μM–10 mM) or clonidine (3–50 mM) were replotted as K_M/V_max versus [I] for cirazoline (a) and clonidine (c), and yielded dissociation constants (abscissal intercepts) for loss of I from EI (in the absence of bound S) of 2.64 mM (cirazoline) and 8.8 mM (clonidine). Thereafter, reciprocals of V_max values were plotted versus [I] for cirazoline (b) and clonidine (d), and yielded dissociation constants (abscissal intercepts) for loss of I from ESI of 14.55 mM (cirazoline) and 270 mM (clonidine). The values for γ, the degree to which the presence of substrate reduces the affinity of enzyme for inhibitor, and thus to which the presence of inhibitor reduces the affinity of enzyme for substrate, were calculated as approximately 5.5 (cirazoline) and 30.7 (clonidine). The analysis was completed on one set of representative data from three similar experiments, and shows the best-fit±s.e. kinetic constants, where standard errors are asymptotic standard errors of best-fit values.

Figure 7

Replots of slopes obtained from Dixon plots (1/v versus [I]) at several substrate concentrations and at high (inhibitory) concentrations of cirazoline and clonidine. Mean velocity data from triplicate determinations, representative of three similar experiments, were plotted on Dixon plots (not shown), and slopes from these plots were replotted versus 1/[S]. Values for K_i and γ were determined by applying the indicated equations. Respective estimates for αK_M and βV_max (the maximally activated K_M and V_max values), calculated by applying percentage values listed in Table 1 to K_M and V_max constants from parallel control data, were 456 μM and 4.834 mOD min⁻¹ (cirazoline), and 804 μM and 7.124 mOD min⁻¹ (clonidine). Calculated K_i and γK_i dissociation constants were 2.85 and 15.95 mM (cirazoline), and 19.1 and 379 mM (clonidine), respectively.

Figure 8

Representative velocity versus [S] data for clonidine (a) and cirazoline (b) obtained as described in the legend to Figure 4, and fitted to an equation describing enzyme activity in the presence of a drug that is a nonessential activator acting at two sites as well as an intersecting linear noncompetitive inhibitor acting at a third site. Data show mean±s.e. mean initial rates from triplicate determinations, where error bars exceed symbol size. While nonlinear regression was successful at all concentrations of clonidine, analyses were unsatisfactory at concentrations of cirazoline below 100 μM, and failed at concentrations below 10 μM (see text). Mean values for variables calculated from these data are listed in Table 2.