Isoprenaline

Study objectives: Previously it has been shown that there is abnormal hormonal control of [Beta.sub.2]-adrenoceptors in asthmatic women. Exogenous progesterone but not estradiol produces paradoxic downregulation and desensitization of [Beta.sub.2]-adrenoceptors in asthmatic women when compared with nonasthmatic subjects. This study investigates the effect of the oral combined contraceptive pill (OCP) on [Beta.sub.2]-adrenoceptor regulation and function in female asthmatic patients.

Patients: The study population was comprised of 11 women with stable mild to moderate asthma. The mean age was 25 years; the [FEV.sub.1] was 89% of predicted, and the forced expiratory flow, mid-expiratory phase ([FEF.sub.25-75%]) was 69% of predicted.

Design: Patients were evaluated while on (day 20 to 21) and off (day 5 to 7) the OCP during a 28-day calendar period.

Measurements: Serum sex hormones, lymphocyte [Beta.sub.2]-adrenoceptor parameters, and bronchodilator and systemic dose-response curves (DRCs) to albuterol (Salbutamol) (100 to 1,600 [mu]g) were measured at both on and off periods.

Results: Serum levels of endogenous estradiol and progesterone were both suppressed by the OCP. Baseline [FEV.sub.1] were not different while patients were on (2.70 L) and off (2.72 L) the OCP. There were no significant differences in lymphocyte [Beta.sub.2]-adrenoceptor parameters between the two phases of the cycle. Receptor density (geometric mean [B.sub.max]) was 1.78 (on OCP) vs 1.86 off OCP) fentomole/[10.sup.6] cells, maximal cyclic adenosine monophosphate response to isoprenaline was 6.60 (on OCP) vs 7.58 (off OCP) pmol/[10.sup.6] cells, and binding affinity was 14.0 (on OCP) and 13.6 (off OCP) pmol/L. Likewise, there were no significant differences in the bronchodilator and systemic DRCs constructed at both phases of the cycle as evaluated: area-under-curve (AUC) [FEV.sub.1] was 0.53 (on OCP) vs 0.56 (off OCP) L.h; and AUC [FEF.sub.25-75%] was 3,130 (on OCP) vs 3,640 (off OCP) L. Potassium (K) and finger tremor responses were unaltered between the two periods: AUC K was 0.50 (on OCP) vs 0.44 (off OCP) mmol.h/L and AUC tremor was 0.72 (on OCP) vs 0.89 (off OCP) log units.h.

Conclusion: The OCP did not alter [Beta.sub.2]-adrenoceptor regulation and function in stable female asthmatic patients. Further studies are required in patients who have premenstrual asthma.

(CHEST 1998; 113:278-82)

Key words: [Beta.sub.2]-adrenoceptor; asthma; oral contraceptive pill

Abbreviations: AUC=area-under-curve; [B.sub.max]_=receptor density; DRC dose-response curve; [E.sub.max]=maximal cyclic adenosine monophosphate response to isoprenaline; [FEF.sub.25-75%]=forced expiratory, flow, mid-expiratory phase; K=potassium; [K.sub.d]=binding affinity; OCP=oral combined contraceptive pill; PBS=phosphate-buffered saline solution

The role of female hormones in the regulation and function of [Beta.sub.2]-adrenoceptors is still unclear. This has obvious implications, particularly in asthma. It has been known for some time now that a proportion of female asthmatic patients suffer premenstrual deterioration of their condition.[1-4] Also, in a few case reports, the treatment with exogenous intramuscular progesterone was reported to prevent the premenstrual deterioration.[5] Previously, studies have shown that in healthy women lymphocyte [Beta.sub.2]-adrenoceptor density and responsiveness are affected by the cyclical influence of ovarian sex hormones.[6] More recently, studies have demonstrated that exogenous progesterone, when given to healthy women during the follicular phase, produced significant upregulation of lymphocyte [Beta.sub.2]-adrenoceptors.[7] However, there appears to be abnormal hormonal regulation of [Beta.sub.2]-adrenoceptors in female patients with asthma. There is a loss of the normal cyclical control of [Beta.sub.2]-adrenoceptor function in female asthmatic patients,[8] and the administration of exogenous progesterone produces a paradoxical downregulation and desensitization of [Beta.sub.2]-adrenoceptors in these patients in contrast to the response in healthy women.[9]

This study was therefore undertaken to investigate whether the oral combined contraceptive pill (OCP) might also have effects on [Beta.sub.2]-adrenoceptor regulation and function in female patients with asthma.

Methods

Subjects

Eleven nonsmoking female subjects whose mean age was 25 years (range, 19 to 40 years) participated in this study. All had long-standing stable asthma of mild to moderate severity previously diagnosed according to American Thoracic Society criteria.[10] None had a history of premenstrual deterioration in asthma control. Mean [FEV.sub.1] was 2.81 L (0.16 SEM), 89.2% (3.6 SEM) of predicted, and the forced expiratory flow, mid-expiratory phase ([FEF.sub.25-75%]) was 2.81 L/s (0.25 SEM), 69.5% (5.6 SEM) of predicted. All gave written informed consent before being entered into the study, which was approved by the Tayside Committee on Medical Research Ethics. A full physical examination and biochemical and hematologic values were within normal limits prior to inclusion. Six subjects were receiving an inhaled corticosteroid (beclomethasone dipropionate or budesonide) in a median dose of 400 [mu]g/d (range, 200 to 1,000 [mu]g/d). All subjects were receiving inhaled B2-agonists on an as-required basis in a dose of [is less than] 200 [mu]g/d. Two subjects were receiving inhaled salmeterol xinofoate, 50 [mu]g bid, and one subject was receiving a sustained-release oral theophylline preparation. All subjects were receiving the 21-day OCP for a period of at least 3 months prior to the study. Eight subjects were receiving monophasic and three subjects were receiving triphasic preparations. Of those receiving monophasic preparations, 7 were receiving ethynyl estradiol, 30 [mu]g/d and one was receiving 35 [mu]g/d of ethynyl estradiol. There were different preparations and doses of progestogens.

Protocol

Subjects were evaluated at two visits in the laboratory, once during the week while off the OCP and once at the end of the 21-day course of the OCP during a 28-day calendar period. At each visit, subjects attended the laboratory at 9 AM, and they withheld short-acting [Beta.sub.2]-agonists for 8 h, long-acting [Beta.sub.2]-agonists for 48 h, and theophylline preparations for 48 h prior to each visit. Inhaled corticosteroids were continued unchanged.

At each visit, an intravenous cannula was inserted into an antecubital vein, and after 30 min rest, blood was removed for determination of serum estradiol and progesterone levels and of lymphocyte [Beta.sub.2]-adrenoceptor values. Then, a dose-response curve (DRC) to albuterol (Salbutamol or Ventolin) inhaled through a metered-dose inhaler via a spacing device Volumatic; Allen& Hanburys; Uxbridge, England) was constructed by giving cumulative doubling doses of 100, 200 (100+100),400 200+200), 800 (400+400) and 1,600 [mu]g (800 [mu]g+800 [mu]g). Measurements of [FEV.sub.1] [FEF.sub.25-75%] plasma K, and postural finger tremor were made at basline and 20 min after each dose increment, with each dose increment given every 30 min.

Measurements

Airway Responses

[FEV.sub.1] and [FEF.sub.25-75%] were measured according to American Thoracic Society criteria[11] with a compact spirometer (Vitalograph; Lenexa, Kan) with a pneumotachograph head and pressure transducer, and on-line computer-assisted determination of [FEV.sub.1] and [FEF.sub.25-75%]. Forced expiratory maneuvers were performed from total lung capacity to residual volume. The best [FEV.sub.1] value was taken from three consistent measurements, and the [FEF.sub.25-75%] was taken from the best test of three consistent forced expiratory curves. A coefficient of variation of less than 3% was considered acceptable.

Systemic Responses

Plasma K was measured by flame photometry (IL943 Analyser; Instrumentation Laboratory; Warrington, England) with analysis done at the end of the study and samples assayed in duplicate. The coefficients of variation for analytical imprecision for intra-and inter-assays were 0.93 and 0.79%, respectively.

Postural finger tremor was measured by a previously validated method using an accelerometer transducer (Entran; Ealing, England). Four recordings were made, and the results stored on a computer for subsequent spectral analysis of total tremor power greater than 2 Hz ([mg.sup.2]/s),with computer-assisted autocovariance. The mean of three consistent recordings was subsequently analyzed.

Lymphocyte [Beta.sub.2]-Adrenoceptor Factors

Receptor density ([B.sub.max]), binding affinity ([K.sub.d]), and maximal cyclic adenosine monophosphate response to isoprenaline ([E.sub.max]) were measured as previously described.[6] In brief, 40 mL whole blood was collected into tubes containing ethylenediamine tetraacetic acid and diluted to 50 mL with phosphate-buffered saline solution (PBS); then, two equal portions were centrifuged with 15 mL a preparation material (Lymphoprep; Nycomed Pharma AS; Oslo, Norway). The lymphocyte layer was removed, and after two further washes with PBS and centrifugation, the lymphocyte pellet was removed and resuspended in 5 mL PBS. Lymphocyte [B.sub.max] and [K.sub.d] were determined with [I.sup.125]-iodocyanopindolol (ICYP; NEN-DuPont [UK], Stevenage, UK) at 8 concentrations between 5 and 160 picomole (pmol)/L. Another substance (CGP12177; Ciba-Geigy; Basel, Switzerland) was added to half the tubes to prevent [I.sup.125]-iodocyanopindolol binding to receptor sites, thus allowing nonspecific binding to be evaluated. Counts were determined with a gamma camera (LKB Wallac; Wallac OY Pharmacia; Turku, Finland), and specific (receptor) binding was calculated from total binding minus nonspecific (nonreceptor) binding. The inter-assay coefficients of variation for analytical imprecision were 10.3% for [B.sub.max] and 5.9% for [K.sub.d]. A radioimmunoassay technique (Incstar; Stillwater, Minn) was used to evaluate cyclic adenosine monophasphate levels following suspension in PBS containing theophylline (100 [mu]M) and bovine serum albumin and stimulation with isoprenaline [10.sup.-4] M([E.sub.max]). The intra- and inter-assay coefficients of variation for analytical imprecision were 2.7 and 10.2%, respectively.

Serum Sex Hormones

Samples for serum estradiol and progesterone were centrifuged at 4 [degrees] C, and the serum was extracted and stored at -20 [degrees] C until they were measured in one batch at the end of the study. Serum estradiol (Sorin Biomedica; Sallugia, Italy) and progesterone (Incstar; Wokingham, UK) were measured by radioimmunoassay. The intra-assay coefficients of variation for analytical imprecision were 2.9 and 3.1% for estradiol and progesterone, respectively.

Statistical analysis

Data for [B.sub.max] were long-transformed prior to analysis as they were not normally distributed. The DRCs were analyzed as maximum delta change from baseline and as area-under-curve (AUC) in order to obviate multiple comparisons at different time points. For all factors, comparisons between visits were made by multifactorial analysis of variance. The power of the study was set at 80% ([Beta] error=0.02) in order to detect a 25% difference in [B.sub.max] and a 20% difference in maximum delta [FEV.sub.1.] A probability value of less than 0.05 (two-tailed test) was considered significant. Data were analyzed using a statistical software package (Statgraphics; C Software Publishing Group; Rockville, Md).

Results

Serum Sex Hormones

Serum estradiol was suppressed due to the effects of the OCP (Table 1). There was a significant (p [is less than] 0.05) difference between the two visits for serum estradiol with levels being higher when patients were off the OCP. Serum progesterone level also was suppressed but was not significantly different between the two visits.

[TABULAR DATA 1 NOT REPRODUCIBLE IN ASCII]

Lymphocyte [Beta.sub.2]-Adrenoceptor Factors

[B.sub.max], [K.sub.d], and [E.sub.max] were not significantly different between the two visits while on and off the OCP (Table 1).

Salbutamol DRCs

Baseline airway and systemic parameters prior to the DRC were comparable between the two visits (Table 1). The albuterol DRCs as maximal delta responses from baseline and as AUC, for both airway and systemic responses, were not significantly different between the two visits, while patients were on and off the OCP (Table 2). Individual values for bronchodilator effects (as [FEV.sub.1] and [FEF.sub.25-75%]) from the DRC are shown in Figure 1. The variability in response was greater for [FEF.sub.25-75%] than for [FEV.sub.1].

[TABULAR DATA 2 NOT REPRODUCIBLE IN ASCII]

Discussion

The results in this study show that the OCP does not affect in-vivo [Beta.sub.2]-adrenoceptor regulation or expression in female asthmatic subjects. Both systemic and bronchodilator DRCs did not show any difference during the cycle. [FEF.sub.25-75%] followed [FEV.sub.1] in showing no difference although, as expected, the variance for the former measurement was somewhat larger. This was in contrast to a previous study which showed that a single 10-mg dose of exogenous medroxyprogesterone acetate produced paradoxical [Beta.sub.2]-adrenoceptor downregulation and desensitization when given during the follicular phase in female asthmatic patients.[9] This latter study would have suggested that there may be diminished [Beta.sub.2]-adrenoceptor mediated responses in female asthmatic subjects exposed to exogenous hormones, in particular progesterone. Whether the lack of effect of the OCP was due to a relatively low dose of the progesterone moiety is unclear, since it is difficult to equate the different preparations and doses of progesterone, as compared with the 10 mg of medroxyprogesterone used in a previous study.[9] In other words, it is conceivable that the downregulating effect of progesterone may be a dose-related phenomenon.

A recent study by Skobeloff et al[12] showed that a higher proportion of hospital admissions of female patients for acute asthma occurred during the perimenstrual period although patients taking the oral contraceptive pill were excluded from this study. Juniper et al[13] reported no change in airway responsiveness to methacholine in female asthmatic subjects with either natural cycles or with the oral contraceptive pill. In previous studies, it appears that there is a cyclical variation in the response to adenosine monophosphate challenge, which is abolished by taking the oral contraceptive pill.[14] Taken together with the findings of the present study, this would therefore suggest that any modulating effects of the oral contraceptive pill on airway hyperreactivity are probably not due to altered [Beta.sub.2]-adrenoceptor function.

The present study has limitations which one may, recognize and accept. Firstly, the power of this study may not have been adequate to detect a change in [B.sub.max] of less than 25%. Secondly, this lack of change in [B.sub.max] may have been due to downregulation of [Beta.sub.2]-adrenoceptors by the prior use of [Beta.sub.2]-agonists. However, [B.sub.max] at both phases of the study was not significantly different from geometric mean values seen in normal subjects (1.91 fentomole (fmol)/[10.sup.6] cells) and asthmatic subjects who were not receiving [Beta.sub.2]-agonists (2.0 fmol/[10.sup.6] cells),[15] indicating that they were not downregulated to start with. Thirdly, there was a heterogeneous selection of exogenous progesterones of different doses which may not have produced a uniform response as observed in the single-dosing study with 10 mg of medroxyprogesterone. Finally, the effects of chronic dosing in these subjects who had been receiving the oral contraceptive for some time may have produced a steady state in the hormone-[Beta.sub.2]-adrenoceptor interaction, whereby a week off the oral contraceptive may be inadequate fir the purposes of a washout. The ideal would have been to evaluate these subjects while they were receiving the oral contraceptive and then to withhold the oral contraceptive for a full cycle and then to evaluate. Unfortunately, this was considered unethical by the hospital medical ethics committee because of the unacceptably high risk of pregnancy in sexually active women.

It is also important to point out that lymphocyte [Beta.sub.2]-adrenoceptors were used as a noninvasive way of following changes in lung [Beta.sub.2]-adrenoceptors, and indeed there is conflicting evidence as to whether this is a valid surrogate.[16-17] In this respect, this study found that lymphocyte [Beta.sub.2]-adrenoceptors mirrored the airway albuterol dose response in that neither showed any difference between the on and off phases of the oral contraceptive pill.

Conclusion

In previous studies, there appears to be an abnormal hormonal regulation of [Beta.sub.2]-adrenoceptors in female asthmatic subjects in response to progesterone. However, this present study appears to show that the OCP does not have any deleterious effect on [Beta.sub.2]-adrenoceptor regulation and function. Further studies are required to examine the effects of the oral contraceptive pill in less stable women who have documented premenstrual asthma.

References

[1] Gibbs CJ, Coutts II, Lock R, et al. Premenstrual exacerbation of asthma. Thorax 1984; 39:833-36

[2] Pauli BD. Reid RL, Mint PW, et al. Influence of the menstrual cycle on airway function in asthmatic and normal subjects. Am Rev Respir Dis 1989; 140:358-62

[3] Hanley SP. Asthma variation with menstruation. Br J Dis Chest 1981; 75:306-08

[4] Eliasson O, Scherzer HH, DeGraff AC. Morbidity in asthma in relation to the menstrual cycle. J Allergy Clin Immunol 1986; 77: 87-94

[5] Benyon HLC, Garbett ND, Barnes PJ Severe premenstrual exacerbations of asthma: effects of intramuscular progesterone. Lancet 1988: 2:370-72

[6] Wheeldon NM, Newnham DM, Coutie WJ, et al. Influence of sex-steroid hormones on the regulation of lymphocyte [Beta.sub.2]-adrenoceptors during the menstrual cycle. Br J Clin Pharmacol 1994; 37:583-88

[7] Tan KS, McFarlane LC, Coutie WJ, et al. Effects of exogenous female sex-steroid hormones on lymphocyte [Beta.sub.2]-adrenoceptors in normal females. Br J Clin Pharmacol 1996; 41: 414-16

[8] Tan KS, McFarlane LC, Lipworth BJ. Loss of normal cyclical [Beta.sub.2]-adrenoceptor regulation and increased premenstrual responsiveness to adenosine monophosphate in stable female asthmatic patients. Thorax 1997; 52:608-11

[9] Tan KS, McFarlane LC, Lipworth BJ. Paradoxical downregulation and desensitization of [Beta.sub.2]-adrenoceptors by exogenous progesterone in female asthmatics. Chest 1997; 111: 847-51

[10] American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease and asthma. Am Rev Respir Dis 1987; 36:225-44

[11] American Thoracic Society. Standardisation of Spirometry 1987 update. Am Rev Respir Dis 1987; 136:1285-98

[12] Skobeloff EM, Spivey WH, Silverman R, et al. The effect of the menstrual cycle on asthma presentations in the emergency department. Arch Intern Med 1996; 156:1837-40

[13] Juniper EF, Kline PA, Roberts RS, et al. Airway responsiveness to methacholine during the natural menstrual cycle and the effect of oral contraceptives. Am Rev Respir Dis, 1987; 135:1039-42

[14] Tan KS, McFarlane LC, Lipworth BJ. Modulation of airway reactivity and peak flow variability in asthmatics receiving the oral contraceptive pill. Am J Respir Crit Care Med 1997; 155:1273-77

[15] Tan KS, Grove A., Cargill RI, et al. Effects of inhaled fluticasone propionate and oral prednisolone on lymphocyte [Beta.sub.2]-adrenoceptor function in asthmatic patients. Chest 1996; 109:343-47

[16] Hauck RW, Bohnn M, Gengenbach PL, et al. Beta-2 adrenoceptors in human lung and peripheral mononuclear leukocytes of terbutaline-treated patients. Chest 1990; 98:376-81

[17] Haves MJ, Qing F, Rhodes CG, et al. In vivo quantification of human pulmonary K2-adrenoceptors: effect of [Beta]-agonist therapy. Am J Respir Crit Care Med 1997; 154:1277-83

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