545

JPP 2003 55 545ndash549szlig 2003 The AuthorsReceived September 20 2002Accepted January 7 2003DOI 101211002235702991ISSN 0022-3573

Department of PharmacologyUniversity of CambridgeTennis Court Road CambridgeCB2 1PD UK

Vandana Jain EmmaMcWilliams Michael Young

Correspondence J M YoungDepartment of PharmacologyUniversity of Cambridge TennisCourt Road Cambridge CB2 1PDUK E-mail jmy1camacuk

Thiomersal enhances the binding of histamine to theH1 receptor but not histamine-stimulated inositolphosphate formation

Vandana Jain Emma McWilliams and Michael Young

Abstract

Thiomersal (thimerosal) was a weak inhibitor of the binding of [3H]mepyramine to histamine H1

receptors in guinea-pig cerebellar membranes (11 sect 1 inhibition at 10 middotM 32 sect 3 inhibition at

300middotM) However in the concentration range 3ndash30 middotM thiomersal enhanced the binding of

histamine to the H1 receptor as reflected by the displacement of curves of histamine inhibition of

[3H]mepyramine binding to lower concentrations without any change in the Hill coefficient The

ratio of the IC50 values (the concentration giving 50 inhibition) in the absence and presence of

thiomersal increased from 18 with 3 middotM to 36 with 30 middotM thiomersal There was no consistent effect

of thiomersal at concentrations of 30 middotM and below on curves of mepyramine inhibition of

[3H]mepyramine binding In the presence of 10 middotM thiomersal histamine-induced accumulation of

inositol phosphates in U373 MG astrocytoma cells was partially inhibited (37 sect 8 inhibition of the

maximum response) without any significant change in the EC50 (the concentration giving the half

maximal response) for histamine Thus although histamine binding was potentiated by thiomersal

there was no potentiation of an H1 receptor-mediated functional response

Introduction

The binding of agonists to the histamine H1 receptor is particularly sensitive toreduction of a dithiol bond by dithiothreitol (DTT) (Donaldson amp Hill 1986a 1987)or to covalent modification of a receptor thiol by N-ethylmaleimide (NEM) (Yeramianet al 1985) whereas the binding of antagonists is relatively little altered by either agent(Yeramian et al 1985 Donaldson amp Hill 1986a Hughes et al 2000) The effect of bothDTT and NEM is to potentiate H1-agonist binding as indicated by a shift to lowerconcentrations of the curve for agonist inhibition of the binding of [3H]mepyramine Inthe case of DTT the potentiation of the binding of H1-agonists can be correlated withan increased potency in functional responses (Fleisch et al 1973 Donaldson amp Hill1986b 1987 Young amp Young 1994) No comparable effect has been demonstratedwith NEM (Fleisch et al 1973) probably as a consequence of additional actions ofNEM at sites post-agonist binding such as the well established inactivation of Gproteins (see Sidhu et al 1991) leading to an overall inhibition of functional responses

The extent to which the action of NEM is general to agents which modify thiols isnot clear In contrast to NEM the Group IIB metal cations Hg2Dagger Cd2Dagger and Zn2Daggerwhich bind strongly to thiols are potent inhibitors of antagonist binding to the H1

receptor particularly Hg2Dagger (Treherne et al 1991) However it is not known whetherorganic mercurials such as thiomersal (thimerosal) behave in the same way asmercuric cations or as an organic thiol reagent

There is also a methodological reason why the action of thiomersal on theH1 receptor is of interest In some studies on the potentiation by thiomersal of 145-IP3-induced Ca2Dagger release from intracellular stores histamine has been used as theagonist to generate 145-IP3 via the activation of H1 receptors (Bootman et al 1992Young et al 1998 Montero et al 2001) It is clear that there is an effect of thiomersal atthe 145-IP3 receptor but the argument has been made that there is no contributionfrom an action at the H1 receptor since 100 middotM thiomersal produced a partial

inhibition of histamine-stimulated accumulation of total[3H]inositol phosphates ([3H]IP) in LiDagger-treated cells(Bootman et al 1992) However the potentiating effectof thiomersal on histamine-induced Ca2Dagger mobilization inU373 MG astrocytoma cells is apparent at a concentra-tion an order of magnitude lower (Young et al 1998) and itis not established whether at this concentration 10 middotMpart of the potentiating effect might not be at the level ofthe H1 receptor We show here that concentrations ofthiomersal in the range 3plusmn30 middotM do enhance the bindingof histamine to the H1 receptor but the effect onhistamine-stimulated [3H]IP accumulation is still partialinhibition

Materials and Methods

Measurement of histamine inhibition of thebinding of [3H]mepyramine to guinea-pigcerebellar membranes

Guinea-pig cerebellar membranes were prepared in Tris-HCl buffer as described previously (Gibson et al 1994)Incubations in 10 mM Tris-HCl buffer pH 75 contained05plusmn075 nM [3H]mepyramine histamine (in a few experi-ments unlabelled mepyramine) thiomersal (where appro-priate) and cerebellar homogenate (approx 02 mgprotein) in a total volume of 1 mL (four replicates ateach histamine concentration eight to twelve replicatesin the absence of inhibitor spread through the experi-ment) Curves in the presence and absence of thiomersalwere always determined in parallel in the same experi-ment Equilibration was for 60 min at 30 macrC and wasterminated by filtration through Whatman GFB glassfibre paper pre-soaked in 03 (wv) polyethyleniminefor 3plusmn5 h using a Brandel (Gaithersburg MD) cellharvester The filters were washed with ice-cold bufferand then transferred to scintillation insert vials containing40 mL scintillator (Emulsifier-Safe Packard) and allowedto stand at room temperature for at least 3 h beforedetermination of tritium by liquid scintillation counting

Histamine-induced [3H]inositol phosphateaccumulation

U373 MG cells (National Culture Collection PortonDown UK) were cultured in Dulbeccorsquos modified Eaglemedium (DMEM)nutrient mixture F-12 (11 vv Gibco)containing 10 foetal calf serum and 2 mM glutamine(Gibco) and supplemented with 1 (vv) anti-mycoticantibiotic mixture (10 000 U mLiexcl1 penicillin 10 mg mLiexcl1

streptomycin and 25 middotg mLiexcl1 amphotericin B Sigma)Cells were dissociated with trypsinEDTA (Sigma)seeded (approx 50 000 cellswell) onto 12-well plates(Costar) and grown to near confluence The culturemedium was removed and the monolayers washed with1 mL inositol-free DMEM before addition of 05 mL inosi-tol-free DMEM containing 10 dialysed calf serum 10middotM myo-inositol and 25 middotCi mLiexcl1 [3H]inositol (016 middotM)

After 20plusmn24 h the labelling medium was aspirated off andthe cells were equilibrated for 10 min at 37 macrC in 10 mLHEPES medium (in mM NaCl 120 KCl 54 MgCl2 16CaCl2 18 HEPES 25 and D-glucose 11 pH 74) This wasremoved HEPES medium containing LiCl (30 mM) added(048 mL) to each well and the cells incubated for 15 min at37 macrC before addition of 20 middotL histamine or histamineDagger 10 middotM thiomersal (four replicates at each histamine con-centration) Cells were then incubated for a further 30 minbefore termination of the reaction by aspirating off themedium rinsing each well with 1 mL ice-cold HEPESbuffer containing LiCl and adding 05 mL 10 per-chloric acid containing 1 mM EDTA and 1 mg mLiexcl1 phyticacid The plates were left to stand on ice for 30 min A 450-middotL sample was taken from each well and added to 400 middotLof a 11 (vv) solution of trioctylamine112-trichlorotri-fluoroethane A portion of the upper phase (035 mL) wastransferred to an insert vial 3 mL 50 mM HEPES bufferpH 76 added and the mixture applied to an AG1 X-8(formate form 100plusmn200 mesh Biorad) anion-exchangecolumn [3H]Inositol and [3H]glycerophosphoinositolwere eluted with 10 mL H2O and 10 mL 60 mM ammo-nium formate5 mM sodium tetraborate respectively and[3H]inositol mono- bis- and tris-phosphates ([3H]IP)eluted with 10 mL 08 M ammonium formate01 M formicacid Quicksafe A (10 mL Zinnser Analytic) was added tothe eluant and the tritium content determined by liquidscintillation counting

Analysis of data

Data for the inhibition of [3H]mepyramine binding byhistamine were fitted by non-linear regression to a Hillequation (logistic equation)

of uninhibited binding of [3H]mepyramine ˆ (100 iexclNS)((AIC50)nH Dagger 1) Dagger NS

where NS is the percentage of the binding of[3H]mepyramine insensitive to inhibition by histamine Ais the concentration of histamine nH is the Hill coefficientand IC50 the concentration of histamine giving 50 inhib-ition of the histamine-sensitive [3H]mepyramine binding

Concentrationplusmnresponse data for histamine-induced[3H]IP accumulation were similarly fitted to a hyperbola

Response ˆ Respmax C(C Dagger EC50)

where Respmax is the maximum response C is the con-centration of histamine and EC50 is the concentrationgiving the half maximal response

Statistical comparison of the IC50 values of curves ofhistamine inhibition of [3H]mepyramine binding in thepresence and absence of thiomersal was made by fittingthe curves simultaneously and assessing the increase in theresidual sum of squares when parameters were con-strained to be the same for both curves (excess sum ofsquares method (Rodbard 1974)) as described by Youngamp Young (1994)

The errors of ratios (eg of uninhibited binding)were calculated using the approximate formula ie the

546 Vandana Jain et al

coefficient of variation of the ratio is equal to the squareroot of the sum of the squares of the coefficients of vari-ation of the numerator and denominator (Colquhoun1971) The overall mean of a series of measurementseach of which provided a mean sect sem was expressedas the weighted mean sect sem (Colquhoun 1971)

Chemicals

[3H]Mepyramine (30 Ci mmoliexcl1) was obtained fromAmersham Biosciences (Little Chalfont BuckinghamshireUK) and [3H]inositol (21 Ci mmoliexcl1) from New EnglandNuclear (Hounslow Middlesex UK) Histamine dihy-drochloride HEPES mepyramine maleate perchloricacid phytic acid thiomersal 112-trichlorotrifluoroethane(freon) tri-n-octylamine and Tris were purchased fromSigma (Poole Dorset UK)

Results and Discussion

Comparison of the effect of thiomersal NEMand HgCl2 on [3H]mepyramine binding

Thiomersal produced only a weak inhibition of the bind-ing of [3H]mepyramine (11 sect 1 inhibition at 10 middotM and32 sect 3 inhibition at 300 middotM) similar to that producedby NEM (Figure 1) In contrast [3H]mepyramine bindingwas potently inhibited by HgCl2 (Figure 1) log(IC50)iexcl537 sect 002 (IC50 43 sect 01 middotM approximate se)consistent with our earlier observations using the sameexperimental protocol for Hg2Dagger IC50 5 middotM (Treherneet al 1991) and NEM (Hughes et al 2000) The very steepcurve for HgCl2 (best-fit Hill slope 269 sect 051) was a

consequence of an effectively irreversible action anddepletion of the free concentration of Hg2Dagger due to exten-sive tissue binding (R Dempster amp J M Young unpub-lished observations)

Effect of thiomersal on histamine bindingto the H1 receptor

The effect of thiomersal 3plusmn30 middotM on the inhibition of thebinding of [3H]mepyramine by histamine was to produce aleftward shift of the inhibition curve to lower concentra-tions without any consistent effect on the Hill slope or theextent of the binding of [3H]mepyramine insensitive toinhibition by histamine (illustrated for 30 middotM thiomersalin Figure 2A) Neither 3 nor 10 middotM thiomersal caused anyleftward shift of curves of the inhibition of the binding of[3H]mepyramine by non-radiolabelled mepyramine (twoexperiments at each concentration data not shown) butin one of two experiments with 30 middotM thiomersal therewas a small 13-fold but statistically significant shiftof the inhibition curve for mepyramine to the right indi-cating a reduction in the affinity of mepyramine Thiseffect was more pronounced with 100 middotM thiomersaland therefore measurements of the effect of thiomersalon the histamine curve were not made at concentrationsgreater than 30 middotM The extent of the shift of the IC50 forhistamine as a function of the concentration of thiomersalis shown in Figure 2B

The extent of the shift of the histamine inhibitioncurves by 10plusmn30 middotM thiomersal was similar to thatproduced by 1plusmn2 mM NEM (Yeramian et al 1985 ourown unpublished observations) In the original experi-ments with NEM (Yeramian et al 1985) as with thosewith DTT (Donaldson amp Hill 1986a) the shift was accom-panied by a decrease in the Hill coefficient which wasinterpreted as an increase in the proportion of a highaffinity state of the receptor This high affinity state hasbeen presumed to represent the ternary complex betweenagonist receptor and G protein and an increase in theamount of this complex would seem likely to be associatedwith an enhanced functional response However it hasbeen questioned whether Hill slopes lt1 for agonist inhib-ition curves are adequately accounted for by the ternarycomplex model particularly with regard to the effects ofmodulators such as NEM (Lee et al 1986) and the obser-vation that there was no significant effect on the slope ofthe histamine inhibition curves in the presence of 3plusmn30 middotM

thiomersal (mean values of the Hill coefficient 077 sect 002in control curves and 079 sect 002 in the presence of1plusmn30 middotM thiomersal six experiments) should be inter-preted with caution with regard to receptor function

Effect of thiomersal on histamine-stimulatedinositol phosphate accumulation

To investigate directly the effect of 10 middotM thiomersal onH1-receptor activation of phospholipase C we measuredhistamine-induced inositol phosphate accumulation inhuman U373 MG astrocytoma cells in the presence and

100

80

60

40

20

0ndash7 ndash6 ndash5 ndash4 ndash3

log [Inhibitor ( )]M

HgCl2

Thiomersal

NEM

o

f u

nin

hib

ited

bin

din

g o

f

[H

]mep

yra

min

e3

Figure 1 Inhibition of the binding of [3H]mepyramine to guinea-pig

cerebellar membranes by thiomersal HgCl2 and NEM Points for

thiomersal are the weighted means sect approximate sem from two to

nine independentmeasurementsThe data for HgCl2 and NEM are from

quadruplicate determinations within a single experiment each of which

was in close agreement with previous observations (Trehene et al 1991

Hughes et al 2000) Where no error bars are apparent the error was

within the size of the symbol The curve drawn for HgCl2 is the best-fit

line to a Hill equation (see Materials and Methods)

Enhanced binding of histamine to the H1 receptor with thiomersal 547

absence of thiomersal Previously we have shown that inthis cell line [3H]IP formation in response to histamineconcentrations of 1 mM and below was mediated by theH1 receptor (Arias-MontanAuml o et al 1994) and that there wasno evidence for the presence of functional H2 receptorswhich might modulate this response (Wong et al 2000)

Histamine alone produced an approximately 20-foldincrease in [3H]IP over a 30-min incubation (Figure 3)This was reduced in the presence of 10 middotM thiomersal

(best-fit values of Responsemax 206- sect 12-fold and129- sect 14-fold stimulation in the absence and presenceof thiomersal respectively) without any significant effecton log(EC50) for histamine iexcl549 sect 012 and iexcl557 sect024 respectively Thiomersal (10 middotM) alone had no con-sistent effect on basal [3H]IP accumulation (570plusmn1040dpm) over the three experiments

Conclusions

Two principle conclusions can be drawn from the dataFirstly thiomersal an organic mercurial which will bindto thiol groups is a weak inhibitor of [3H]mepyraminebinding but enhances the binding of histamine to the H1

receptor In this respect thiomersal acts in the same wayas NEM and not as the free Hg2Dagger ion Potentiation of H1-agonist binding may thus be an effect shared by organicthiol reagents Secondly the enhanced binding in the pre-sence of 10 middotM thiomersal is not reflected in any increase inhistamine-induced [3H]IP accumulation on the contrarythere is an inhibition of the maximum response This mayreflect a second action of thiomersal at the level of Gprotein coupling analogous to the action of NEM or ata post-receptor site

References

Arias-MontanAuml o J-A Berger V Young J M (1994) Calcium-dependence of histamine- and carbachol-induced inositolphosphate formation in human U373 MG astrocytoma cellscomparison with HeLa cells and brain slices Br J Pharmacol111 598plusmn608

100

80

60

40

20

0ndash7 ndash6 ndash5 ndash4 ndash3

log [histamine ( )]M

+ 30thiomersal

mM

Control

o

f u

nin

hib

ited

bin

din

g

of

[H

]mep

yram

ine

3

ndash8

4

3

2

1

00 10 20 30

[Thiomersal] ( )mM

IC50

his

tam

ine

con

tro

lIC

50+

thio

mer

sal

A B

Figure 2 Enhancement by thiomersal of the binding of histamine to the histamine H1 receptor A Inhibition by histamine of the binding of

061nM [3H]mepyramine in the presence or absence of 30 middotM thiomersal Points are the means sect approximate sem from quadruplicate

determinations within a single experiment Where no error bars are apparent the error was within the size of the symbol The curves drawn are

the best-fit lines to a Hill equation (see Materials and Methods) Best-fit parameters sect estimated se in the absence and presence of thiomersal

Hill coefficient 077sect 003 and 077 sect003 IC50 337sect 016 and 104sect 006middotM non-specific binding 131sect 06 and 135sect 05 respectively

B Concentration dependence of the extent of the enhance of histamine binding by thiomersal Points are the ratios of the best-fit IC50 values

for histamine inhibition of [3H]mepyramine binding in the absence and presence of thiomersal sect approximate se taken from independent

experiments (such as that shown in A above)All shifts were statistically significant as assessed by the excess sum of squares test (see Analysis of

data) except in one experiment with 1 middotM thiomersal The curve drawn is a best-fit polynomial and is solely to emphasize the trend of the data

2500

2000

1500

1000

500

0ndash7 ndash6 ndash5 ndash4 ndash3

log [histamine ( )]M

Control

+ 10 thiomersalmM[H

]IP

accu

mu

late

d(

of

bas

al)

3

Figure 3 Effect of 10 middotM thiomersal on histamine stimulated [3H]IP

accumulation in monolayers of U373 MG astrocytoma cells Values

are the weighted meanssect se from three independent experiments

(one to three determinations at each concentration) Where no error

bars are apparent the error was within the size of the symbol The

curve drawn is the best-fit line to a hyperbola (best-fit values of EC50

and Bmax are given in the text)

548 Vandana Jain et al

inhibition of histamine-stimulated accumulation of total[3H]inositol phosphates ([3H]IP) in LiDagger-treated cells(Bootman et al 1992) However the potentiating effectof thiomersal on histamine-induced Ca2Dagger mobilization inU373 MG astrocytoma cells is apparent at a concentra-tion an order of magnitude lower (Young et al 1998) and itis not established whether at this concentration 10 middotMpart of the potentiating effect might not be at the level ofthe H1 receptor We show here that concentrations ofthiomersal in the range 3plusmn30 middotM do enhance the bindingof histamine to the H1 receptor but the effect onhistamine-stimulated [3H]IP accumulation is still partialinhibition

Materials and Methods

Measurement of histamine inhibition of thebinding of [3H]mepyramine to guinea-pigcerebellar membranes

Guinea-pig cerebellar membranes were prepared in Tris-HCl buffer as described previously (Gibson et al 1994)Incubations in 10 mM Tris-HCl buffer pH 75 contained05plusmn075 nM [3H]mepyramine histamine (in a few experi-ments unlabelled mepyramine) thiomersal (where appro-priate) and cerebellar homogenate (approx 02 mgprotein) in a total volume of 1 mL (four replicates ateach histamine concentration eight to twelve replicatesin the absence of inhibitor spread through the experi-ment) Curves in the presence and absence of thiomersalwere always determined in parallel in the same experi-ment Equilibration was for 60 min at 30 macrC and wasterminated by filtration through Whatman GFB glassfibre paper pre-soaked in 03 (wv) polyethyleniminefor 3plusmn5 h using a Brandel (Gaithersburg MD) cellharvester The filters were washed with ice-cold bufferand then transferred to scintillation insert vials containing40 mL scintillator (Emulsifier-Safe Packard) and allowedto stand at room temperature for at least 3 h beforedetermination of tritium by liquid scintillation counting

Histamine-induced [3H]inositol phosphateaccumulation

U373 MG cells (National Culture Collection PortonDown UK) were cultured in Dulbeccorsquos modified Eaglemedium (DMEM)nutrient mixture F-12 (11 vv Gibco)containing 10 foetal calf serum and 2 mM glutamine(Gibco) and supplemented with 1 (vv) anti-mycoticantibiotic mixture (10 000 U mLiexcl1 penicillin 10 mg mLiexcl1

streptomycin and 25 middotg mLiexcl1 amphotericin B Sigma)Cells were dissociated with trypsinEDTA (Sigma)seeded (approx 50 000 cellswell) onto 12-well plates(Costar) and grown to near confluence The culturemedium was removed and the monolayers washed with1 mL inositol-free DMEM before addition of 05 mL inosi-tol-free DMEM containing 10 dialysed calf serum 10middotM myo-inositol and 25 middotCi mLiexcl1 [3H]inositol (016 middotM)

After 20plusmn24 h the labelling medium was aspirated off andthe cells were equilibrated for 10 min at 37 macrC in 10 mLHEPES medium (in mM NaCl 120 KCl 54 MgCl2 16CaCl2 18 HEPES 25 and D-glucose 11 pH 74) This wasremoved HEPES medium containing LiCl (30 mM) added(048 mL) to each well and the cells incubated for 15 min at37 macrC before addition of 20 middotL histamine or histamineDagger 10 middotM thiomersal (four replicates at each histamine con-centration) Cells were then incubated for a further 30 minbefore termination of the reaction by aspirating off themedium rinsing each well with 1 mL ice-cold HEPESbuffer containing LiCl and adding 05 mL 10 per-chloric acid containing 1 mM EDTA and 1 mg mLiexcl1 phyticacid The plates were left to stand on ice for 30 min A 450-middotL sample was taken from each well and added to 400 middotLof a 11 (vv) solution of trioctylamine112-trichlorotri-fluoroethane A portion of the upper phase (035 mL) wastransferred to an insert vial 3 mL 50 mM HEPES bufferpH 76 added and the mixture applied to an AG1 X-8(formate form 100plusmn200 mesh Biorad) anion-exchangecolumn [3H]Inositol and [3H]glycerophosphoinositolwere eluted with 10 mL H2O and 10 mL 60 mM ammo-nium formate5 mM sodium tetraborate respectively and[3H]inositol mono- bis- and tris-phosphates ([3H]IP)eluted with 10 mL 08 M ammonium formate01 M formicacid Quicksafe A (10 mL Zinnser Analytic) was added tothe eluant and the tritium content determined by liquidscintillation counting

Analysis of data

Data for the inhibition of [3H]mepyramine binding byhistamine were fitted by non-linear regression to a Hillequation (logistic equation)

of uninhibited binding of [3H]mepyramine ˆ (100 iexclNS)((AIC50)nH Dagger 1) Dagger NS

where NS is the percentage of the binding of[3H]mepyramine insensitive to inhibition by histamine Ais the concentration of histamine nH is the Hill coefficientand IC50 the concentration of histamine giving 50 inhib-ition of the histamine-sensitive [3H]mepyramine binding

Concentrationplusmnresponse data for histamine-induced[3H]IP accumulation were similarly fitted to a hyperbola

Response ˆ Respmax C(C Dagger EC50)

where Respmax is the maximum response C is the con-centration of histamine and EC50 is the concentrationgiving the half maximal response

Statistical comparison of the IC50 values of curves ofhistamine inhibition of [3H]mepyramine binding in thepresence and absence of thiomersal was made by fittingthe curves simultaneously and assessing the increase in theresidual sum of squares when parameters were con-strained to be the same for both curves (excess sum ofsquares method (Rodbard 1974)) as described by Youngamp Young (1994)

The errors of ratios (eg of uninhibited binding)were calculated using the approximate formula ie the

546 Vandana Jain et al

coefficient of variation of the ratio is equal to the squareroot of the sum of the squares of the coefficients of vari-ation of the numerator and denominator (Colquhoun1971) The overall mean of a series of measurementseach of which provided a mean sect sem was expressedas the weighted mean sect sem (Colquhoun 1971)

Chemicals

[3H]Mepyramine (30 Ci mmoliexcl1) was obtained fromAmersham Biosciences (Little Chalfont BuckinghamshireUK) and [3H]inositol (21 Ci mmoliexcl1) from New EnglandNuclear (Hounslow Middlesex UK) Histamine dihy-drochloride HEPES mepyramine maleate perchloricacid phytic acid thiomersal 112-trichlorotrifluoroethane(freon) tri-n-octylamine and Tris were purchased fromSigma (Poole Dorset UK)

Results and Discussion

Comparison of the effect of thiomersal NEMand HgCl2 on [3H]mepyramine binding

Thiomersal produced only a weak inhibition of the bind-ing of [3H]mepyramine (11 sect 1 inhibition at 10 middotM and32 sect 3 inhibition at 300 middotM) similar to that producedby NEM (Figure 1) In contrast [3H]mepyramine bindingwas potently inhibited by HgCl2 (Figure 1) log(IC50)iexcl537 sect 002 (IC50 43 sect 01 middotM approximate se)consistent with our earlier observations using the sameexperimental protocol for Hg2Dagger IC50 5 middotM (Treherneet al 1991) and NEM (Hughes et al 2000) The very steepcurve for HgCl2 (best-fit Hill slope 269 sect 051) was a

consequence of an effectively irreversible action anddepletion of the free concentration of Hg2Dagger due to exten-sive tissue binding (R Dempster amp J M Young unpub-lished observations)

Effect of thiomersal on histamine bindingto the H1 receptor

The effect of thiomersal 3plusmn30 middotM on the inhibition of thebinding of [3H]mepyramine by histamine was to produce aleftward shift of the inhibition curve to lower concentra-tions without any consistent effect on the Hill slope or theextent of the binding of [3H]mepyramine insensitive toinhibition by histamine (illustrated for 30 middotM thiomersalin Figure 2A) Neither 3 nor 10 middotM thiomersal caused anyleftward shift of curves of the inhibition of the binding of[3H]mepyramine by non-radiolabelled mepyramine (twoexperiments at each concentration data not shown) butin one of two experiments with 30 middotM thiomersal therewas a small 13-fold but statistically significant shiftof the inhibition curve for mepyramine to the right indi-cating a reduction in the affinity of mepyramine Thiseffect was more pronounced with 100 middotM thiomersaland therefore measurements of the effect of thiomersalon the histamine curve were not made at concentrationsgreater than 30 middotM The extent of the shift of the IC50 forhistamine as a function of the concentration of thiomersalis shown in Figure 2B

The extent of the shift of the histamine inhibitioncurves by 10plusmn30 middotM thiomersal was similar to thatproduced by 1plusmn2 mM NEM (Yeramian et al 1985 ourown unpublished observations) In the original experi-ments with NEM (Yeramian et al 1985) as with thosewith DTT (Donaldson amp Hill 1986a) the shift was accom-panied by a decrease in the Hill coefficient which wasinterpreted as an increase in the proportion of a highaffinity state of the receptor This high affinity state hasbeen presumed to represent the ternary complex betweenagonist receptor and G protein and an increase in theamount of this complex would seem likely to be associatedwith an enhanced functional response However it hasbeen questioned whether Hill slopes lt1 for agonist inhib-ition curves are adequately accounted for by the ternarycomplex model particularly with regard to the effects ofmodulators such as NEM (Lee et al 1986) and the obser-vation that there was no significant effect on the slope ofthe histamine inhibition curves in the presence of 3plusmn30 middotM

thiomersal (mean values of the Hill coefficient 077 sect 002in control curves and 079 sect 002 in the presence of1plusmn30 middotM thiomersal six experiments) should be inter-preted with caution with regard to receptor function

Effect of thiomersal on histamine-stimulatedinositol phosphate accumulation

To investigate directly the effect of 10 middotM thiomersal onH1-receptor activation of phospholipase C we measuredhistamine-induced inositol phosphate accumulation inhuman U373 MG astrocytoma cells in the presence and

100

80

60

40

20

0ndash7 ndash6 ndash5 ndash4 ndash3

log [Inhibitor ( )]M

HgCl2

Thiomersal

NEM

o

f u

nin

hib

ited

bin

din

g o

f

[H

]mep

yra

min

e3

Figure 1 Inhibition of the binding of [3H]mepyramine to guinea-pig

cerebellar membranes by thiomersal HgCl2 and NEM Points for

thiomersal are the weighted means sect approximate sem from two to

nine independentmeasurementsThe data for HgCl2 and NEM are from

quadruplicate determinations within a single experiment each of which

was in close agreement with previous observations (Trehene et al 1991

Hughes et al 2000) Where no error bars are apparent the error was

within the size of the symbol The curve drawn for HgCl2 is the best-fit

line to a Hill equation (see Materials and Methods)

Enhanced binding of histamine to the H1 receptor with thiomersal 547

absence of thiomersal Previously we have shown that inthis cell line [3H]IP formation in response to histamineconcentrations of 1 mM and below was mediated by theH1 receptor (Arias-MontanAuml o et al 1994) and that there wasno evidence for the presence of functional H2 receptorswhich might modulate this response (Wong et al 2000)

Histamine alone produced an approximately 20-foldincrease in [3H]IP over a 30-min incubation (Figure 3)This was reduced in the presence of 10 middotM thiomersal

(best-fit values of Responsemax 206- sect 12-fold and129- sect 14-fold stimulation in the absence and presenceof thiomersal respectively) without any significant effecton log(EC50) for histamine iexcl549 sect 012 and iexcl557 sect024 respectively Thiomersal (10 middotM) alone had no con-sistent effect on basal [3H]IP accumulation (570plusmn1040dpm) over the three experiments

Conclusions

Two principle conclusions can be drawn from the dataFirstly thiomersal an organic mercurial which will bindto thiol groups is a weak inhibitor of [3H]mepyraminebinding but enhances the binding of histamine to the H1

receptor In this respect thiomersal acts in the same wayas NEM and not as the free Hg2Dagger ion Potentiation of H1-agonist binding may thus be an effect shared by organicthiol reagents Secondly the enhanced binding in the pre-sence of 10 middotM thiomersal is not reflected in any increase inhistamine-induced [3H]IP accumulation on the contrarythere is an inhibition of the maximum response This mayreflect a second action of thiomersal at the level of Gprotein coupling analogous to the action of NEM or ata post-receptor site

References

Arias-MontanAuml o J-A Berger V Young J M (1994) Calcium-dependence of histamine- and carbachol-induced inositolphosphate formation in human U373 MG astrocytoma cellscomparison with HeLa cells and brain slices Br J Pharmacol111 598plusmn608

100

80

60

40

20

0ndash7 ndash6 ndash5 ndash4 ndash3

log [histamine ( )]M

+ 30thiomersal

mM

Control

o

f u

nin

hib

ited

bin

din

g

of

[H

]mep

yram

ine

3

ndash8

4

3

2

1

00 10 20 30

[Thiomersal] ( )mM

IC50

his

tam

ine

con

tro

lIC

50+

thio

mer

sal

A B

Figure 2 Enhancement by thiomersal of the binding of histamine to the histamine H1 receptor A Inhibition by histamine of the binding of

061nM [3H]mepyramine in the presence or absence of 30 middotM thiomersal Points are the means sect approximate sem from quadruplicate

determinations within a single experiment Where no error bars are apparent the error was within the size of the symbol The curves drawn are

the best-fit lines to a Hill equation (see Materials and Methods) Best-fit parameters sect estimated se in the absence and presence of thiomersal

Hill coefficient 077sect 003 and 077 sect003 IC50 337sect 016 and 104sect 006middotM non-specific binding 131sect 06 and 135sect 05 respectively

B Concentration dependence of the extent of the enhance of histamine binding by thiomersal Points are the ratios of the best-fit IC50 values

for histamine inhibition of [3H]mepyramine binding in the absence and presence of thiomersal sect approximate se taken from independent

experiments (such as that shown in A above)All shifts were statistically significant as assessed by the excess sum of squares test (see Analysis of

data) except in one experiment with 1 middotM thiomersal The curve drawn is a best-fit polynomial and is solely to emphasize the trend of the data

2500

2000

1500

1000

500

0ndash7 ndash6 ndash5 ndash4 ndash3

log [histamine ( )]M

Control

+ 10 thiomersalmM[H

]IP

accu

mu

late

d(

of

bas

al)

3

Figure 3 Effect of 10 middotM thiomersal on histamine stimulated [3H]IP

accumulation in monolayers of U373 MG astrocytoma cells Values

are the weighted meanssect se from three independent experiments

(one to three determinations at each concentration) Where no error

bars are apparent the error was within the size of the symbol The

curve drawn is the best-fit line to a hyperbola (best-fit values of EC50

and Bmax are given in the text)

548 Vandana Jain et al

coefficient of variation of the ratio is equal to the squareroot of the sum of the squares of the coefficients of vari-ation of the numerator and denominator (Colquhoun1971) The overall mean of a series of measurementseach of which provided a mean sect sem was expressedas the weighted mean sect sem (Colquhoun 1971)

Chemicals

[3H]Mepyramine (30 Ci mmoliexcl1) was obtained fromAmersham Biosciences (Little Chalfont BuckinghamshireUK) and [3H]inositol (21 Ci mmoliexcl1) from New EnglandNuclear (Hounslow Middlesex UK) Histamine dihy-drochloride HEPES mepyramine maleate perchloricacid phytic acid thiomersal 112-trichlorotrifluoroethane(freon) tri-n-octylamine and Tris were purchased fromSigma (Poole Dorset UK)

Results and Discussion

Comparison of the effect of thiomersal NEMand HgCl2 on [3H]mepyramine binding

Thiomersal produced only a weak inhibition of the bind-ing of [3H]mepyramine (11 sect 1 inhibition at 10 middotM and32 sect 3 inhibition at 300 middotM) similar to that producedby NEM (Figure 1) In contrast [3H]mepyramine bindingwas potently inhibited by HgCl2 (Figure 1) log(IC50)iexcl537 sect 002 (IC50 43 sect 01 middotM approximate se)consistent with our earlier observations using the sameexperimental protocol for Hg2Dagger IC50 5 middotM (Treherneet al 1991) and NEM (Hughes et al 2000) The very steepcurve for HgCl2 (best-fit Hill slope 269 sect 051) was a

consequence of an effectively irreversible action anddepletion of the free concentration of Hg2Dagger due to exten-sive tissue binding (R Dempster amp J M Young unpub-lished observations)

Effect of thiomersal on histamine bindingto the H1 receptor

The effect of thiomersal 3plusmn30 middotM on the inhibition of thebinding of [3H]mepyramine by histamine was to produce aleftward shift of the inhibition curve to lower concentra-tions without any consistent effect on the Hill slope or theextent of the binding of [3H]mepyramine insensitive toinhibition by histamine (illustrated for 30 middotM thiomersalin Figure 2A) Neither 3 nor 10 middotM thiomersal caused anyleftward shift of curves of the inhibition of the binding of[3H]mepyramine by non-radiolabelled mepyramine (twoexperiments at each concentration data not shown) butin one of two experiments with 30 middotM thiomersal therewas a small 13-fold but statistically significant shiftof the inhibition curve for mepyramine to the right indi-cating a reduction in the affinity of mepyramine Thiseffect was more pronounced with 100 middotM thiomersaland therefore measurements of the effect of thiomersalon the histamine curve were not made at concentrationsgreater than 30 middotM The extent of the shift of the IC50 forhistamine as a function of the concentration of thiomersalis shown in Figure 2B

The extent of the shift of the histamine inhibitioncurves by 10plusmn30 middotM thiomersal was similar to thatproduced by 1plusmn2 mM NEM (Yeramian et al 1985 ourown unpublished observations) In the original experi-ments with NEM (Yeramian et al 1985) as with thosewith DTT (Donaldson amp Hill 1986a) the shift was accom-panied by a decrease in the Hill coefficient which wasinterpreted as an increase in the proportion of a highaffinity state of the receptor This high affinity state hasbeen presumed to represent the ternary complex betweenagonist receptor and G protein and an increase in theamount of this complex would seem likely to be associatedwith an enhanced functional response However it hasbeen questioned whether Hill slopes lt1 for agonist inhib-ition curves are adequately accounted for by the ternarycomplex model particularly with regard to the effects ofmodulators such as NEM (Lee et al 1986) and the obser-vation that there was no significant effect on the slope ofthe histamine inhibition curves in the presence of 3plusmn30 middotM

thiomersal (mean values of the Hill coefficient 077 sect 002in control curves and 079 sect 002 in the presence of1plusmn30 middotM thiomersal six experiments) should be inter-preted with caution with regard to receptor function

Effect of thiomersal on histamine-stimulatedinositol phosphate accumulation

To investigate directly the effect of 10 middotM thiomersal onH1-receptor activation of phospholipase C we measuredhistamine-induced inositol phosphate accumulation inhuman U373 MG astrocytoma cells in the presence and

100

80

60

40

20

0ndash7 ndash6 ndash5 ndash4 ndash3

log [Inhibitor ( )]M

HgCl2

Thiomersal

NEM

o

f u

nin

hib

ited

bin

din

g o

f

[H

]mep

yra

min

e3

Figure 1 Inhibition of the binding of [3H]mepyramine to guinea-pig

cerebellar membranes by thiomersal HgCl2 and NEM Points for

thiomersal are the weighted means sect approximate sem from two to

nine independentmeasurementsThe data for HgCl2 and NEM are from

quadruplicate determinations within a single experiment each of which

was in close agreement with previous observations (Trehene et al 1991

Hughes et al 2000) Where no error bars are apparent the error was

within the size of the symbol The curve drawn for HgCl2 is the best-fit

line to a Hill equation (see Materials and Methods)

Enhanced binding of histamine to the H1 receptor with thiomersal 547

absence of thiomersal Previously we have shown that inthis cell line [3H]IP formation in response to histamineconcentrations of 1 mM and below was mediated by theH1 receptor (Arias-MontanAuml o et al 1994) and that there wasno evidence for the presence of functional H2 receptorswhich might modulate this response (Wong et al 2000)

Histamine alone produced an approximately 20-foldincrease in [3H]IP over a 30-min incubation (Figure 3)This was reduced in the presence of 10 middotM thiomersal

(best-fit values of Responsemax 206- sect 12-fold and129- sect 14-fold stimulation in the absence and presenceof thiomersal respectively) without any significant effecton log(EC50) for histamine iexcl549 sect 012 and iexcl557 sect024 respectively Thiomersal (10 middotM) alone had no con-sistent effect on basal [3H]IP accumulation (570plusmn1040dpm) over the three experiments

Conclusions

Two principle conclusions can be drawn from the dataFirstly thiomersal an organic mercurial which will bindto thiol groups is a weak inhibitor of [3H]mepyraminebinding but enhances the binding of histamine to the H1

receptor In this respect thiomersal acts in the same wayas NEM and not as the free Hg2Dagger ion Potentiation of H1-agonist binding may thus be an effect shared by organicthiol reagents Secondly the enhanced binding in the pre-sence of 10 middotM thiomersal is not reflected in any increase inhistamine-induced [3H]IP accumulation on the contrarythere is an inhibition of the maximum response This mayreflect a second action of thiomersal at the level of Gprotein coupling analogous to the action of NEM or ata post-receptor site

References

Arias-MontanAuml o J-A Berger V Young J M (1994) Calcium-dependence of histamine- and carbachol-induced inositolphosphate formation in human U373 MG astrocytoma cellscomparison with HeLa cells and brain slices Br J Pharmacol111 598plusmn608

100

80

60

40

20

0ndash7 ndash6 ndash5 ndash4 ndash3

log [histamine ( )]M

+ 30thiomersal

mM

Control

o

f u

nin

hib

ited

bin

din

g

of

[H

]mep

yram

ine

3

ndash8

4

3

2

1

00 10 20 30

[Thiomersal] ( )mM

IC50

his

tam

ine

con

tro

lIC

50+

thio

mer

sal

A B

Figure 2 Enhancement by thiomersal of the binding of histamine to the histamine H1 receptor A Inhibition by histamine of the binding of

061nM [3H]mepyramine in the presence or absence of 30 middotM thiomersal Points are the means sect approximate sem from quadruplicate

determinations within a single experiment Where no error bars are apparent the error was within the size of the symbol The curves drawn are

the best-fit lines to a Hill equation (see Materials and Methods) Best-fit parameters sect estimated se in the absence and presence of thiomersal

Hill coefficient 077sect 003 and 077 sect003 IC50 337sect 016 and 104sect 006middotM non-specific binding 131sect 06 and 135sect 05 respectively

B Concentration dependence of the extent of the enhance of histamine binding by thiomersal Points are the ratios of the best-fit IC50 values

for histamine inhibition of [3H]mepyramine binding in the absence and presence of thiomersal sect approximate se taken from independent

experiments (such as that shown in A above)All shifts were statistically significant as assessed by the excess sum of squares test (see Analysis of

data) except in one experiment with 1 middotM thiomersal The curve drawn is a best-fit polynomial and is solely to emphasize the trend of the data

2500

2000

1500

1000

500

0ndash7 ndash6 ndash5 ndash4 ndash3

log [histamine ( )]M

Control

+ 10 thiomersalmM[H

]IP

accu

mu

late

d(

of

bas

al)

3

Figure 3 Effect of 10 middotM thiomersal on histamine stimulated [3H]IP

accumulation in monolayers of U373 MG astrocytoma cells Values

are the weighted meanssect se from three independent experiments

(one to three determinations at each concentration) Where no error

bars are apparent the error was within the size of the symbol The

curve drawn is the best-fit line to a hyperbola (best-fit values of EC50

and Bmax are given in the text)

548 Vandana Jain et al

absence of thiomersal Previously we have shown that inthis cell line [3H]IP formation in response to histamineconcentrations of 1 mM and below was mediated by theH1 receptor (Arias-MontanAuml o et al 1994) and that there wasno evidence for the presence of functional H2 receptorswhich might modulate this response (Wong et al 2000)

Histamine alone produced an approximately 20-foldincrease in [3H]IP over a 30-min incubation (Figure 3)This was reduced in the presence of 10 middotM thiomersal

(best-fit values of Responsemax 206- sect 12-fold and129- sect 14-fold stimulation in the absence and presenceof thiomersal respectively) without any significant effecton log(EC50) for histamine iexcl549 sect 012 and iexcl557 sect024 respectively Thiomersal (10 middotM) alone had no con-sistent effect on basal [3H]IP accumulation (570plusmn1040dpm) over the three experiments

Conclusions

Two principle conclusions can be drawn from the dataFirstly thiomersal an organic mercurial which will bindto thiol groups is a weak inhibitor of [3H]mepyraminebinding but enhances the binding of histamine to the H1

receptor In this respect thiomersal acts in the same wayas NEM and not as the free Hg2Dagger ion Potentiation of H1-agonist binding may thus be an effect shared by organicthiol reagents Secondly the enhanced binding in the pre-sence of 10 middotM thiomersal is not reflected in any increase inhistamine-induced [3H]IP accumulation on the contrarythere is an inhibition of the maximum response This mayreflect a second action of thiomersal at the level of Gprotein coupling analogous to the action of NEM or ata post-receptor site

References

Arias-MontanAuml o J-A Berger V Young J M (1994) Calcium-dependence of histamine- and carbachol-induced inositolphosphate formation in human U373 MG astrocytoma cellscomparison with HeLa cells and brain slices Br J Pharmacol111 598plusmn608

100

80

60

40

20

0ndash7 ndash6 ndash5 ndash4 ndash3

log [histamine ( )]M

+ 30thiomersal

mM

Control

o

f u

nin

hib

ited

bin

din

g

of

[H

]mep

yram

ine

3

ndash8

4

3

2

1

00 10 20 30

[Thiomersal] ( )mM

IC50

his

tam

ine

con

tro

lIC

50+

thio

mer

sal

A B

Figure 2 Enhancement by thiomersal of the binding of histamine to the histamine H1 receptor A Inhibition by histamine of the binding of

061nM [3H]mepyramine in the presence or absence of 30 middotM thiomersal Points are the means sect approximate sem from quadruplicate

determinations within a single experiment Where no error bars are apparent the error was within the size of the symbol The curves drawn are

the best-fit lines to a Hill equation (see Materials and Methods) Best-fit parameters sect estimated se in the absence and presence of thiomersal

Hill coefficient 077sect 003 and 077 sect003 IC50 337sect 016 and 104sect 006middotM non-specific binding 131sect 06 and 135sect 05 respectively

B Concentration dependence of the extent of the enhance of histamine binding by thiomersal Points are the ratios of the best-fit IC50 values

for histamine inhibition of [3H]mepyramine binding in the absence and presence of thiomersal sect approximate se taken from independent

experiments (such as that shown in A above)All shifts were statistically significant as assessed by the excess sum of squares test (see Analysis of

data) except in one experiment with 1 middotM thiomersal The curve drawn is a best-fit polynomial and is solely to emphasize the trend of the data

2500

2000

1500

1000

500

0ndash7 ndash6 ndash5 ndash4 ndash3

log [histamine ( )]M

Control

+ 10 thiomersalmM[H

]IP

accu

mu

late

d(

of

bas

al)

3

Figure 3 Effect of 10 middotM thiomersal on histamine stimulated [3H]IP

accumulation in monolayers of U373 MG astrocytoma cells Values

are the weighted meanssect se from three independent experiments

(one to three determinations at each concentration) Where no error

bars are apparent the error was within the size of the symbol The

curve drawn is the best-fit line to a hyperbola (best-fit values of EC50

and Bmax are given in the text)

548 Vandana Jain et al

Bootman M D Taylor C W Berridge M J (1992) The thiolreagent thimerosal evokes Ca2Dagger spikes in HeLa cells by sen-sitising the inositol 145-trisphosphate receptor J Biol Chem267 25113plusmn25119

Colquhoun D (1971) Lectures on biostatistics Clarendon PressOxford pp 24 39

Donaldson J Hill S J (1986a) 14-Dithiothreitol-inducedalteration in histamine H1-agonist binding in guinea-pig cere-bellum and cerebral cortex Eur J Pharmacol 129 25plusmn31

Donaldson J Hill S J (1986b) Enhancement of histamineH1-receptor agonist activity by 14-dithiothreitol in guinea-pig cerebellum and cerebral cortex J Neurochem 471476plusmn1482

Donaldson J Hill S J (1987) 14-Dithiothreitol-inducedchanges in histamine H1-agonist efficacy and affinity in thelongitudinal smooth muscle of guinea-pig ileum Br JPharmacol 90 263plusmn271

Fleisch J H Krzan M C Titus E (1973) Pharmacologicreceptor activity of rabbit aorta effect of dithiothreitol andN-ethylmaleimide Circ Res 33 284plusmn290

Gibson W J Roques T W Young J M (1994) Modulationof antagonist binding to histamine H1-receptors by sodiumions and by 2-amino-2-hydroxymethyl-propan-13-diol HClBr J Pharmacol 111 1262plusmn1268

Hughes S-A C F Gibson W J Young J M (2000) Theinteraction of U-73122 with the histamine H1-receptor impli-cations for the use of U-73122 in defining H1 receptor-coupledsignalling pathways Naunyn Schmiedebergs Arch Pharmacol362 555plusmn558

Lee T W T Sole M J Wells J W (1986) Assessment of aternary model for the binding of agonists to neurohumoralreceptors Biochemistry 25 7009plusmn7020

Montero M Barrero M J Torrecilla F Lobaton C DMoreno A Alvarez J (2001) Stimulation by thimerosal ofhistamine-induced Ca2Dagger release in intact HeLa cells seen withaequorin targeted to the endoplasmic reticulum Cell Calcium30 181plusmn190

Rodbard D (1974) Statistical quality control and routine dataprocessing for radioimmunoassays and immunoradiometricassays Clin Chem 20 1255plusmn1270

Sidhu A Sullivan M Kohout T Balen P Fishman P H(1991) D1Dopamine receptors can interact with both stimula-tory and inhibitory guanine nucleotide binding proteins JNeurochem 57 1445plusmn1451

Treherne J M Stern J S Flack W J Young J M (1991)Inhibition by cations of antagonist binding to histamine H1-receptors differential effect of sodium ions on the binding oftwo radioligands Br J Pharmacol 103 1745plusmn1751

Wong M-P M Cooper D M F Young K W Young J M(2000) Characteristicsof theCa2Dagger-dependentinhibitionof cyclicAMP accumulation by histamine and thapsigargin in humanU373 MG astrocytoma cells Br J Pharmacol 130 1021plusmn1030

Yeramian E Garbarg M Schwartz J-C (1985) N-Ethylmaleimide-induced changes in agonist affinity for hista-mine H1-receptors in the guinea-pig brain Mol Pharmacol28 155plusmn162

Young K W Young J M (1994) Potentiation by 14-dithio-threitol of histamine-induced inositol phosphate formation inrat cerebral cortex and human HeLa cells Eur J Pharmacol269 283plusmn292

Young K W Pinnock R D Nahorski S R (1998)Determination of the inositol (145) trisphosphate requirementfor histamine- and substance P-induced Ca2Dagger mobilisation inhuman U373 MG astrocytoma cells Cell Calcium 24 59plusmn70

Enhanced binding of histamine to the H1 receptor with thiomersal 549