European Journal of Pharmacology - Molecular Pharmacology Section, 247 (1993) 347-351 347 Elsevier Science Publishers B.V.

EJPMOL 90549

Short communication

Mg 2÷ modulates the binding of [3H]glibenclamide to its receptor in rat cerebral cortical membranes

Kevin L e e and Michae l L.J. A sh fo rd *

Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1Q J, UK

Received 14 July 1993; revised MS received 27 August 1993; accepted 31 August 1993

Radioligand binding studies were performed to determine the effect of various cations on the characteristics of [3H]glibenclamide binding to its high-affinity receptor in rat cerebral cortex membranes. Mg 2+ was shown specifically to reduce radioligand binding to membranes pretreated with 5 mM EDTA. CaCI 2 enhanced [3H]glibenclamide binding whilst MnCI2, KC1 and NaCI were without significant effect. 2 mM MgC12 induced a statistically significant rightward shift in the dissociation constant for glibenclamide obtained from both saturation and competition studies. These results suggest that Mg 2+ may participate in the regulation of the sulphonylurea receptor in the rat cerebral cortex.

Mg2+; Sulphonylureas; KAX P channels

1. Introduction

The sulphonylureas are a group of drugs which have been shown to inhibit the ATP sensitive potassium (KAT P) channel (Sturgess et al., 1985). High- and low- affinity binding sites for glibenclamide, the most potent sulphonylurea, have been identified in a number of tissues including brain, pancreatic /3 cells, smooth and cardiac tissue (for review see Ashcroft and Ashcroft, 1992). The localisation of these receptors closely paral- lels the distribution of the KAT P channel itself which has led to the suggestion that the sulphonylurea recep- tor is an integral component of this ion channel.

Although the sulphonylurea receptor and KAT P channel are closely coupled in the pancreatic /3 cell, the linkage between channel and receptor appears less close in neuronal tissue (Ashford et al., 1990). Re- cently, we have shown that the sulphonylurea receptor and KAT P channel can be functionally uncoupled in the CRI G1 insulin-secreting cell line by removal of intra- cellular Mg 2÷ (Lee and Ashford, 1993), although this process does not affect the affinity or number of recep- tors for glibenclamide in this cell line (Ozanne and Hales, personal communication).

In the present study, we have examined the effects of various divalent and monovalent cations on gliben- clamide binding to its high-affinity receptor in rat

Corresponding author. Tel.: (0223) 334000; Fax: (0223) 334040.

cerebral cortical membranes. In contrast to pancreatic /3 cell membranes, we now report that Mg 2+ can modulate the binding characteristics of glibenclamide to its high-affinity binding site when steps are taken to remove any residual bound divalent cations.

2. Materials and methods

Rat brains were removed and immediately im- mersed in ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM EDTA. The cerebral cortex was dissected out and homogenised in 10 volumes of the same buffer using a glass homogeniser with motor- driven pestle (five up and down strokes at 3000 rpm). The homogenate was then centrifuged at 20,000 × g for 30 min at 4°C. The resultant pellet was washed and re-suspended in ice-cold 50 mM Tris-HC1 buffer (pH 7.4), but this time in the absence of EDTA, and cen- trifuged again at 20,000 × g for 30 min at 4°C. Finally, the pellet was re-suspended in 50 mM Tris-HC1 buffer to give a final protein concentration of 2 mg /ml . The membrane suspension was kept on ice and used the same day. Protein concentration was determined es- sentially as described by Lowry et al. (1951) using bovine serum albumin as standard.

Incubations were carried out in 50 mM Tris-HC1 buffer (pH 7.4) containing [3H]glibenclamide (50.9 C i / mmol; NEN, Boston, MA, USA), competitor (where appropriate) and cerebral cortex membrane ho- mogenate (0.4 mg protein) in a total volume of 0.5 ml.

348

Equilibration was for 2 h at 25°C and was terminated by addition of 4 ml ice-cold buffer. The mixture was immediately filtered through Whatman G F / B glass fibre filters using a Brandel (Gaithersburg, MD, USA) 24-place cell harvester. The filters were washed (3 × 4 ml) with ice-cold buffer and transferred to scintillation vials and treated with 4 ml of scintillant (Emulsifier- safe, Packard, Downers Grove, IL, USA). After stand- ing overnight, the radioactivity was determined by liq- uid scintillation counting. Non-specific binding of radi- oligand was determined by undertaking parallel incu- bations in the presence of 1 ~M glibenclamide or glipizide.

Saturation curves for the amount of specific binding of [3H]glibenclamide versus its concentration were fit- ted to the Hill equation;

Bmax'[L] n [3H]glibenclamide bound

[L] n + EC~0

where Bma x is the maximum amount of [3H]glibencla- mide bound; [L] is the concentration of [3H]- glibenclamide; n is the Hill coefficient; and ECs0 is the concentration of [3H]glibenclamide at which the amount of [3H]glibenclamide bound is 50% of Bma x

Curves of inhibition of [3H]glibenclamide binding were fitted to the equation;

% of uninhibited binding 1 0 0 - NS + N S

of [3H]glibenclamide - ( [A] / ICs0) n + 1

where [A] is the concentration of inhibitor; IC50 the inhibitor concentration giving 50% inhibition of the inhibitor sensitive binding; and NS the percentage of [3H]glibenclamide binding insensitive to the inhibitor.

The inhibition constants (K i) for unlabelled ligands were calculated from the concentration dependence of the inhibition of [3H]glibenclamide binding by the method of Cheng and Prusoff (1973), using the follow- ing equation;

IC5o Ki

1 + ([L]/Ka)

where K a is the dissociation constant for gliben- clamide.

The data were fitted to the appropriate equations by weighting the points with the variance associated with them and performing non-linear regression analysis using either GraphPAD In Plot (GraphPAD Software, San Diego, CA, USA) as implemented on the XENi 286/45 (Apricot computers, Birmingham, UK) or the Harwell library routine VBO1A as implemented on the Cambridge IBM 3081/3084.

In experiments where the effect of Mg 2÷ or Mn 2+ on the parameters associated with [3H]glibenclamide binding was determined, statistical comparisons were made by fitting the curves obtained in the presence and absence of cation simultaneously (by using the

NAG library routine E04FDF) and assessing the resid- ual sum of squares when one of the parameters (e.g., EC50) was constrained to be the same for both curves by calculating the F-statistic (Rodbard, 1974). Data are presented in the text as the arithmetic mean value + S.E.M.

50 mM Tris-HCl buffer was made up in ultra-pure water obtained from a Milli Q Plus Reagent Grade Water Purification System (Millipore, Watford, UK) and was kept at 4°C until required. Stock solutions of all drugs and inorganic salts used in the binding assay were obtained from Sigma Chemicals (St. Louis, MO, USA) and were made up freshly on the day of the experiment. Glipizide was donated by Pfizer (Sand- wich, Kent).

(a)

200

15()

V 100 g

V 5o

0 -6

i i i | i

-5 -4 -3 -2 -f Log (Cation Concn, M)

(b)

2OO

150

~ 100

,~ 50

O' -6

i i i i i

-5 -4 -3 -2 -1 Log (Cation Concn, M)

Fig. 1. The effect of monovalent and divalent cations on the binding of [3H]glibenclamide to rat cerebral cortex membranes. (A) Increas- ing concentrations of NaCI (o ) and KCI ( n ) were incubated with [3H]glibenclamide (0.25 to 0.4 nM) for 2 h at 25°C in the presence of rat cerebral cortex membranes (0.8 m g / m l ) in a total assay volume of 0.5 ml. Each curve is a representative example of four experiments. Each point is the mean of four replicate determinations with the standard error of the mean shown by vertical bars; where no error bars are shown, the error was within the size of the symbol. (B) Increasing concentrations of MgCI 2 (e), CaCI 2 ( • ) and MnC1 a ( • ) were incubated with [3H]glibenclamide (0.25 to 0.4 nM) for 2 h at 25°C in the presence of rat cerebral cortex membranes (0.8 m g / m l ) in a total assay volume of 0.5 ml. Each curve is a representative example of four (CaCI 2 and MnCI 2) or seven (MgCI 2) experiments. Each point is the mean of four replicate determinations with the s tandard error of the mean shown by vertical bars; where no error

bars are shown, the error was within the size of the symbol.

3. Results

In order to examine the effect of Mg 2+ and other cations on [3H]glibenclamide binding, the homogenate was initially centrifuged in 5 mM E D T A containing 50 mM Tris-HC1 buffer in an attempt to remove any residual bound cations that may have been present during homogenate preparation.

Preliminary experiments (not shown) produced evi- dence that the characteristics of [3H]glibenclamide binding were not affected by this procedure. For exam- ple, dissociation and association rate constants of 0.0057 + 0.0013 min - t (n = 6) and 3.37 x 10 7 min -1

(a)

(b)

(c)

Z

c~

100 ~

80

60

40

20

0 - 1 2 I i I I

- 11 - 1 0 - 9 - 8

Log (Glibenclamide, M)

~ 1 5 0 0

lOOO oo

• < 5 0 0

0 -10 .5

©

- 10.0 -9 .5 -9 .0 -8 .5

Log (3H-Gtibenclamide concn, M)

1 5 0 0

@

1ooo (Z3

<E 5oo

0 -10 .5

0 o

- 10 .0 -9 .5 -9 .0 - 8 5

Log (3H-Glibenclamide concn, M)

349

M - l (n = 6) were obtained from kinetic studies. From these two values a dissociation constant (K d) of 0.17 nM was calculated and an optimum incubation time of 2 h obtained. Specific binding of [3H]glibenclamide to a fixed concentration of membrane protein (0.8 m g / m l ) represented 50-70% of the total binding and was to a single site with a K d of 0.71 + 0.22 nM (n = 10) and a Bma x of 82.60 + 12.20 p m o l /g (n = 10). The Hill coeffi- cients for the fitted curves did not significantly differ from one; 1.16 + 0.29 (n = 10; Fig. 2b). Fig. 2a demon- strates that increasing concentrations of unlabelled glibenclamide inhibited the binding of [3H]glibencla- mide to its high-affinity site. From these studies, a dissociation constant of 0.57 + 0.12 nM (n = 8) was obtained. The above values are very similar to those previously obtained without an initial ED TA wash procedure (Ashcroft and Ashcroft, 1992). Thus these results would suggest that this pretreatment procedure does not alter [3H]glibenclamide binding characteris- tics per se.

As shown in previous studies (Gopalakrishnan et al., 1991), increasing concentrations of the monovalent

Fig. 2. The effect of 2 mM MgC1 a and 2 mM MnCI a on the binding characteristics of [3H]glibenclamide to rat cerebral cortex mem- branes (0.8 mg /ml ) . (A) The effect of 2 mM MgCI 2 on the inhibition of [3H]glibenclamide binding by unlabelled glibenclamide. Increasing concentrations of glibenclamide were incubated with [3H]glibencla- mide (0.35 nM) and rat cerebral cortex membranes at 25°C for 2 h in the absence (open circles) or presence (closed circles) of 2 m M MgCI 2. Semi logarithmic plot of one representative experiment with each point representing the mean of four replicate determinations with the s tandard error of the mean shown by vertical bars. The presence of 2 m M MgCI 2 caused a statistically significant shift in the K d for glibenclamide from 0.58 nM to 4.58 nM (P < 0.01). The Hill coefficients were not statistically different; 1.03+0.30 (no MgCI 2) and 0.76+_0.15 (with MgCI2), nor were the levels of non-specific binding; 24.2 +_ 3.7% and 14.3 +_ 11.2%, respectively. (B) The effect of 2 m M MgCI 2 on the binding of increasing concentrations of [3H]glibenclamide to rat cerebral cortex membranes . Incubations were performed in a final volume of 0.5 ml at 25°C for 2 h in the presence (closed circles) or absence (open circles) of 2 mM MgCI e. Semi logarithmic plot of one representative experiment with each point represent ing the mean of four replicate determinat ions with the s tandard error of the mean shown by vertical bars. The presence of 2 mM MgCI 2 caused a statistically significant shift in the K d from 0.49 nM to 3.00 nM (P < 0.01). The Bma x values were not statisti- cally different; 1790 + 159 dpm (no MgCI z) and 2230 +- 223 dpm (with MgCIe), nor were the associated Hill coefficients; 1.32+-0.24 and 0.95+_0.10 respectively. (C) The effect of 2 mM MnCI 2 on the binding of increasing concentrat ions of [3H]glibenclamide to rat cerebral cortex membranes . Incubations were performed in a final volume of 0.5 ml at 25°C for 2 h in the presence (closed circles) or absence (open circles) of 2 mM MnCI 2. Semi logarithmic plot of one representative experiment with each point representing the mean of four replicate determinations with the s tandard error of the mean shown by vertical bars. The presence of 2 mM MnC12 did not statistically alter the value of the K d (0.88 +_ 0.15 nM and 1.08 +_ 0.33 nM in the absence or presence of 2 mM MnCI2), Bma x (1790+_ 120 dpm and 1850 +_ 212 dpm, respectively) or Hill coefficient (1.21 +_ 0.19

and 1.10 + 0.25, respectively).

350

cations NaCI (n = 4) and KC1 (n = 4) were found not to affect the characteristics of [3H]glibenclamide bind- ing (Fig. la) over the concentration range tested (10 /zM to 100 mM). In contrast in 7 out of 11 experiments conducted over the same concentration range, MgC12 produced a clear displacement of radiolabel such that 100 mM MgC12 induced approximately 70 + 6% inhibi- tion of specific [3H]glibenclamide binding. The g i for this effect was 1.26 + 0.59 mM (Fig. lb). A similar effect was observed when the sulphate salt was em- ployed (n = 3 out of 5 experiments). In those experi- ments where displacement of radiolabel was not ob- served, a small enhancement of [3H]glibenclamide binding was seen (not shown). A similar enhancement of radioligand binding was also observed when similar studies were performed on membranes not subjected to pretreatment with EDTA (n = 2).

In order to examine the specificity of this effect, the divalent cation salts CaC12 and MnC12 were also tested. As shown in Fig. lb, CaCI 2 was found to enhance [3H]glibenclamide binding such that in the presence of 100 mM CaCI2, the amount of [3H]glibenclamide bound was 175% that bound in the absence of the salt. Fig. lb also illustrates that MnC12 did not appear to exert any effect on radioligand binding over this concentration range.

In an attempt to further investigate the effect of MgC12 upon the binding characteristics of gliben- clamide, saturation and inhibition studies were per- formed both in the presence and absence of 2 mM MgCI 2. As illustrated in Fig. 2a, the addition of this divalent cation produced a statistically significant right- ward shift in the Kd obtained from competition studies from 0.57 + 0.12 nM (n = 8) to 4.83 + 1.67 nM (n = 4; P < 0.01). At these concentrations, the addition of MgCI 2 did not affect the Hill coefficient or the level of non-specific binding. Similarly, 2 mM MgC12 also in- duced a significant shift (P<0 .01) in the Kd for [3H]glibenclamide obtained by saturation studies. In this instance, the K d was shifted from 0.71 + 0.22 nM (n = 10) to 3.20 + 0.43 nM (n = 3) by the addition of this cation (Fig. 2b). No change in the values for the Hill coefficient or Bma x were observed.

In contrast, 2 mM MnCI2 did not significantly affect the binding of [3H]glibenclamide in three experiments (Fig. 2c).

4. Discussion

These studies suggest that the divalent cation Mg 2+ is capable of specifically interacting with, and thereby modulating the binding of [3H]glibenclamide to its receptor in rat cerebral cortex membranes. Although this study has not considered the mechanism of this process, the ability of both chloride and sulphate salts

of Mg 2+ to produce this effect, and also the inability of other divalent cations to induce similar responses sug- gests that this action is specific to Mg 2+.

This is the first time this phenomenon has been reported and conflicts with lack of effect of Mg e+ reported by Gopalakrishnan et al. (1991). In another study, Zini et al. (1993) have reported that Mg 2+ does not alter the binding of glibenclamide to its high-affin- ity site but reduces the affinity of the low-affinity receptor. The disparity between these reports may be due to differences in the method of membrane prepa- ration between groups. Both Zini et al. and Gopalakr- ishnan et al. did not appear to pretreat the membranes with EDTA in an attempt to remove residual bound cations. Although this procedure in itself does not appear to affect the binding characteristics of gliben- clamide, it appears to facilitate the ability of Mg 2+ to interact with the radioligands receptor. The reasons for this are unclear, perhaps EDTA removes another bound cation to allow Mg 2+ to interact with this bind- ing site and allosterically alter the receptors affinity for glibenclamide. Previous studies have shown there to be important sulphydryl groups associated with this recep- tor (Gopalakrishnan et al., 1991). It is possible there- fore that this pretreatment process may, by removing certain cations, stabilise the redox state of these func- tions (Torchinsky, 1981) which may in itself facilitate the interaction between cation and receptor. Alterna- tively the observed differences in results may have arisen from differences in the way membranes are stored. We have observed subtle changes in the affinity and number of [3H]glibenclamide receptors in cerebral cortex membranes which have been subjected to freez- ing (Lee and Ashford, unreported observations). Con- sequently, in this study only freshly prepared mem- branes have been used.

In the experiments where Mg 2+ failed to reduce radioligand binding, an enhancement was observed similar to that reported previously in cardiac mem- branes by French et al. (1991). In the present study Mg 2+ enhanced glibenclamide binding consistently in membranes not prewashed with EDTA and a similar effect was also induced by CaCl 2. Thus it may be that this enhancement of binding is a relatively non-specific divalent cation effect and that EDTA failed to remove the divalent cations responsible and hence, stabilise the sulphonylurea receptor in the experiments where this was seen. Studies performed on pancreatic /3 cell membranes and neuronal homogenates regarding the importance of Mg 2+ in mediating the ability of nu- cleotides such as ADP and ATP to alter [3H]glibenclamide binding characteristics have also led to conflicting results. For example Niki et al. (1990) reported that ADP in the presence of Ca 2+ could specifically displace [3H]glibenclamide but ATP was ineffective. Schwanstecher et al. (1991) however, pro-

vide evidence to suggest that ATP and not ADP can specifically inhibit [3H]glibenclamide binding in the presence of Mg e÷. It is possible that these differences could have arisen for reasons such as those proposed above.

The physiological significance of these observations are difficult to interpret. Although the levels of Mg 2+ in the cell would be close to the K i of 1.26 + 0.59 mM reported here (Lennard and Singh, 1991), most of this would be bound to nucleotides such as ATP (Corkey et al., 1987). Finally, these results underline further the differences which exist between the sulphonylurea re- ceptor present in central neurones and the pancreatic /3 cell and in conjunction with previous studies (Lee and Ashford, 1993; Ashford et al., 1990) provide addi- tional evidence that the coupling between sulphony- lurea receptor and KAT e channel in the two tissues is also different.

Acknowledgements

We would like to thank Mrs. W.J. Gibson for technical assistance and Dr J.M. Young for his advice regarding data analysis. K.L. is a Wellcome prize student.

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