Pergamon 0306-3623(94)00136-7

Gen. Pharmac. Vol. 25, No. 8. pp. 1607-1610, 1994 Copyright © 1994 Elsevier Science Ltd

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Increased Affinity of Histamine H 1 Binding to Membranes of Human Myometrium at

the End of Pregnancy R O D R I G O G O N Z A L E Z , E N R I Q U E E C H E V E R R I A , K A R I N R E I N I C K E

and M. I S O L D E R U D O L P H * Departamento de Farmacologia, Facultad de Ciencias Biol6gicas, Universidad de Concepci6n, casilla 152-C,

Concepcidn, Chile [Fax: 56-41-240280]

(Received 30 March 1994)

Abstract--1. The characterization of H I binding sites in membrane preparations of human myometrium obtained from pregnant and non-pregnant women was performed by using 3H-mepyramine as the radioactive iigand.

2. Saturation curve analysis revealed that 3H-mepyramine is bound to a single class of binding sites. Changes in the H~ site binding parameters were observed at the end of pregnancy, resulting in an increased affinity relative to non-pregnant tissue (Kd: 131.0 + 8.8 (non-pregnant) and 72.5 + 7.5 (pregnant) nM, n = 6, P < 0.01).

3. A reduction in receptor concentration at the end of pregnancy was also observed, [Bmx: 565.2 + 43.7 (non-pregnant) and 309.6 +25.9 (pregnant) fmol/mg prot, n =6, P <0.01]. It is possible that this reduction in Bm~ could be attributed to a dilution factor due to the increase in membraneous proteins that occurs during gestation.

Key Words: Histamine H t receptor, pregnancy, human myometrium, mepyramine, astemizole

INTRODUCTION

Histamine is an important intercellular mediator in several tissues. It is released from histamine-storing cells and regulates cellular function via histamine receptors on the effector cell surface. In the mam- malian reproductive system, histamine is stored in mast cells, and its release is regulated by sexual hormones (Sandvei et al., 1986; Crow et al., 1988; Padilla et al., 1990). From studies in rodents, there is increasing evidence that uterine histamine plays a significant role in blastocyst implantation, uterine tissue growth, and as a local mediator of the actions of estrogens in the uterus, such as vasodilatation and edema (Spaziani, 1975; Brandon and Wallis, 1977; Befus, 1990). Furthermore, increasing concentrations of histamine have been demonstrated in the mouse uterus as pregnancy progresses, especially during the second half of pregnancy, followed by a drastic reduction some hours after labor, suggesting a massive release of this amine during the process of parturition (Padilla et al., 1990).

*To whom all correspondence should be addressed.

Histamine has recently been postulated as a mediator in the regulation of human myometrium contractility, since it has been shown to stimulate uterine contractions of/n vitro preparations of human myometrial tissue (Cruz et al., 1989). This effect is mediated through the activation of H~ receptors and increases toward the end of gestation (Cruz et al.,

1989). Besides this, it is potentiated by threshold con- centrations of both serotonin and PGF2~ (Rudolph et al., 1993). The greater contractile activity of the myometrium, in response to histamine at the end of gestation, could be attributed to specific changes in histamine receptor parameters (K d or Bm~), or to an unspecific facilitatory effect for the multiple agents which can cause myometrial contractions, or both. Elucidation of this mechanism could be important in the evaluation of a possible physiological or patho- physiological role of histamine in labor.

During the last two decades, receptors for several neurotransmitters have been identified and character- ized by means o f / n vitro binding studies in mem- branes isolated from different tissues. As a rule, the Hm receptors from different organs in various animal species revealed similar binding characteristics,

1607

1608 RODRIGO GONZALEZ et al.

3H-mepyramine being the most used ligand (Hill, 1990). At low concentrations, mepyramine has been shown to act as a selective competitive antagonist of histamine contractile action in different smooth muscle cells (Mitsuhashi and Payan, 1988; Cruz et al., 1989; Dickenson and Hill, 1992).

The present study characterizes H~ histamine bind- ing sites in membranes of human myometrium using mepyramine as the radioactive ligand. The exper- iment is designed to determine whether pregnancy has any effect on H~ receptor binding parameters that could explain the greater sensitivity of uterine tissue toward histamine at the end of gestation. We report that pregnancy results in an increase in the affinity of H~ binding sites.

METHODS

Consent was obtained from subjects to obtain biopsies of myometrium at the Hospital Higueras, Talcahuano (Chile). Tissue from pregnant patients was obtained from the upper transverse incisional edge of the lower uterine segment at the time of term elective cesarea. Tissue from non-pregnant patients was obtained from uteri which had been submitted for diagnostic histopathology. The clinical and pathological findings of the cases were adenomyosis and leiomyomas.

Preparation o f uterine membranes

Uterine biopsies, kept frozen for 24hr, were weighed and homogenized in 30 volumes of ice-cold buffer Tris-HC1 50 mM, pH 7.4, using a Waring Blendor at maximum velocity for 65 sec. The hom- ogenates were centrifuged at 850g for 10 min at 4°C, the supernatants were filtered through double-layered gauze cloth and recentrifuged at 31,000 g for 20 min at 4°C. The supernatant was discarded and the membrane pellets were resuspended with ultraturrax in 100 volumes of ice-cold Tris-HC1 50 mM, pH 7.4 and centrifuged again at 31,000g for 20 min. This whole procedure was repeated and the final mem- brane pellet was dispersed with the ultraturrax in suitable volumes of Tris-HCl 50 mM, pH 7.4, result- ing in a final protein concentration of 0.6 mg/ml.

Receptor binding assay

Because 3H-histamine exhibited a high degree of non-specific binding for H 1 receptors, 3H-mepyra- mine, the most commonly used 3H-ligand for these receptors (Hill, 1990) was chosen for binding assays. All experiments were performed using disposable polystyrene tubes. Triplicate samples of the mem- brane suspension containing 150 ~g of protein were preincubated for 2 min at 30°C, with or without the

addition of drugs and non-radioactive displacers, in tubes containing Tris-HCI 50mM, pH 7.4 in a final volume of 250 #l. Following preincubation, 3H-mepyramine was added to the tubes and the incubation continued for an additional 30 min. The binding assay was terminated by adding 5 ml of ice-cold buffer to the tubes and rapidly filtering under reduced pressure through polyethylenimine pre- soaked Whatman GF/C glass filters as described previously (Mitsuhashi and Payan, 1988). The filters containing the membrane-bound 3H-mepyramine were then washed twice with 5 ml of ice-cold Tris- HCl buffer. The whole procedure of filtering lasted no longer than 8 sec. The GF/C filters were then trans- ferred to scintilation vials containing 10ml of a Toluene counting solution: PPO (2g/l), POPOP (0.7 g/l) and Triton X-100 (30% v/v). The scintilation vials were agitated during 3 hr and analyzed for tritium in a Beckmann LS-100 Scintillation counter with a counting efficiency of 54%.

To determine the dissociation constant (Kd) and the receptor concentration (Bmax), experiments were performed with six concentrations of 3H-mepyra- mine, from 5 to 180 nM. Under these conditions, specific binding was defined as the difference between total binding in the presence of the radioligand alone and the binding measured when 5 #M mepyramine was added. Competition experiments were performed with 120 nM 3H-mepyramine and increasing concen- trations of competitors. Protein was measured using the method of Lowry et al. (1951) with bovine albumin as the standard.

Determination o f binding parameters and ICso values

Saturation curves for 3H-mepyramine were sub- jected to Scatchard analysis (Scatchard, 1949) and to a Hill plot (Bennett, 1978). Binding parameters (Kd, Bm~x and Hill slope) were calculated by using a computational program (EBDA, G. A. McPherson, Elsevier-Biosoft, 1985). The ICs0 values were deter- mined by log-logit graph analysis (Rodbard and Frazier, 1975).

Chemicals

3H-mepyramine (23.9 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA, U.S.A.). Histamine, structural analogs and other drugs and chemicals were obtained from Sigma Chemical Co. St Louis, MO, U.S.A. Astemizole was a gift from Laboratorios Andrrmaco S.A., Chile.

RESULTS

Preliminary experiments were performed in order to obtain the optimum binding conditions of 3H-

Histamine receptors in human myometrium 1609

mepyramine to human uterine membranes. The maximum specific binding of 3H-mepyramine w as

obtained at 30°C and at pH 7.4. The equilibrium was reached at 25 min and this remained constant for at least 50 min. Under these experimental con- ditions, the specific binding of 3H-mepyramine was linear with protein concentration ranging from 1 0 0 - 1 0 0 0 # g /ml .

The ability of unlabeled histamine, mepyramine and astemizole to compete with radioactive mepyra- mine for the binding to H l sites was analyzed in uterine membranes from pregnant patients. Studies performed with 120 nM 3H-mepyramine and increas- ing concentrations of unlabeled histamine, mepyra- mine or astemizole showed that these compounds competed for the binding sites according to an inhibi- tory pattern, suggesting a single class of HI binding sites. ICs0 values (mean _ SEM, n = 3) for histamine, mepyramine and astemizole, at a range of con- centrations between l n M and l m M were 200.7 _ 15.4 nM, 83.2 _+ 2.5 nM and 67 _+ 5.8 nM, re- spectively (Fig. 1). Other endogenous compounds and drugs such as serotonin, acetylcholine, nor- epinephrine, cimetidine, thioperamide, nicotine, at- ropine, isoproterenol and propanolol did not displace the binding of 3H-mepyramine at concentrations up to 10#M.

Scatchard plots revealed that 3H-mepyramine binds to a single class of binding sites (Fig. 2). Binding parameters of 3H-mepyramine to the H l binding site of uterine membranes from pregnant and

100 ~ ~

" i 80

'~ 60 g o

" 20

g" =

0 -8 -7 -6 -5

Ligand concentration (M)

Fig. 1. Pharmacological characteristics of the H I binding sites in isolated human myometrial membranes. Binding was inhibited by astemizole (O) (ICs0: 67.0+ 5.SnM), mepyramine (*) (ICs0:83.2 + 2.5 nM) and histamine ( × ) (ICs0:200.7 + 15.4 nM). Histamine H 2 or H3 antagonists: cimetidine (Q) or thioperamide (&), which were unable to

displace the radioactive ligand 3H-mepyramine.

. \ \ \

I I INNNN I I 1 " 4 I 0 4(1 80 120 t60 20t) 240 280 320 360 401)

[Boundl pM

Fig. 2. Pharmacological characteristics of the H~ binding sites in isolated human myometrial membranes. Seatchard plot of 3H-mepyramine binding depicted from one exper- iment from (x) pregnant and (0) non-pregnant women. Both fit best to a single line, suggesting a single class of binding sites with different values for Ka and Bm~ as shown

in Table 1.

non-pregnant patients are shown in Table 1. Both Ka and Bmx were reduced significantly in the pregnant condition. No changes were observed in Hill coefficient.

DISCUSSION

Our binding studies of mepyramine to histamine receptors in human myometrial membranes showed the presence of a single class of binding sites. Binding at these sites could be displaced by histamine, mepyramine and astemizole in a concentration- dependent manner, as expected for the Hi receptor, while the H2 and H3 receptor antagonists, cimetidine and thioperamide, did not displace the binding of 3H-mepyramine at concentrations up to 10 #M.

The dissociation constant for 3H-mepyramine binding, obtained in human myometrial membranes, was higher than that observed in other membrane preparations, namely, 0.77 nM for guinea-pig urinary bladder (Kondo et al., 1985) and 3.4 nM in Hela cells (Arias and Young, 1993) but similar to that reported in a cultured smooth muscle cell line (219 and 279nM) (Mitsuhashi and Payan, 1988; 1989) and a clonal Hela cell line (164nM) (Raymond et al., 1991), where binding was correlated to a functional Hi-receptor response. Mepyramine binding Kd values (pregnant: 72.5 + 7.7 riM; non-pregnant: 131.0+8.8nM), agree reasonably well with the mepyramine concentration needed to inhibit uterine contractions evoked by histamine in isolated human uterine myometrial strips (>10nM) (Cruz et al., 1989). This suggests that the characterized histamine H~ binding site in human myometrial membranes could correspond fairly well with a functional H~

GP 2518---F

1610 RODRIGO GONZALEZ et aL

Table 1. Characteristics of the specific binding of 3H-mepyramine to the H i histamine binding sites in human myometrial membranes: the effect of pregnancy

Bmax Kd Experimental condition (fmol/mg prot.) (nM) Hill slope

Non-pregnant 565.2 + 43.7 131.0 + 8.8 0.998 Pregnant 309.6 + 25.9* 72.5 +_ 7.5t 1.000

Membranes from human uteri were incubated for 30 min at 30"C in Tris-HC150 mM pH 7.4 with increasing concentrations of 3H-mepyramine (5-180 nM) and 5 # M mepyramine. Bin, x, K a and Hill slope were calculated by using a computational program as described in the Methods Section. The results are the means + SEM of six independent experiments (from different patients), each one performed in triplicate.

*P <0.001 and fP < 0.01 respective to uterine membranes from non-pregnant women.

receptor tha t cont r ibutes to elicit uterine contrac-

tions. An addi t ional a rgument favoring the hypoth-

esis of a funct ional H~ binding site, is the reduct ion

in the Kd observed at the end of pregnancy (Table 1).

This result could explain the increase in the sensitivity

o f the myomet r ium to the contract i le effects of

his tamine. The reduct ion in Bmax observed at the end

of pregnancy, could be a t t r ibuted to a di lut ion factor

due to the increment in m em br aneous proteins tha t

occurs in different m a m m a l i a n species, as pregnancy

progresses (Kyozuka et al., 1988).

His tamine H~ responses may be i m p o r t a n t in

several aspects of obstetric physiology and pa tho-

physiology including control of uterine flow

(Spaziani, 1975), blastocyst implan ta t ion (Brandon

and Wallis, 1977), ini t ia t ion of par tur i t ion (Padilla

et aL, 1990) and changes in uterine responsiveness in

systemic allergic react ions (Klein et al., 1984). The

ability to determine changes in H~ receptor affinity

and concen t ra t ion as par t of these processes could

give a bet ter comprehens ion of the problem.

Acknowledgements--This work was supported by research grants from FONDECYT (91-0365), Direceirn de Inves- tigacirn, Universidad de Concepcirn (93-3368) and Labora- torios Andrrmaco S. A. Chile. We thank Dr Peter Ward for helpful revision of the manuscript.

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