help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Engelhardt, S.
Right arrow Articles by Brodde, O.-E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Engelhardt, S.
Right arrow Articles by Brodde, O.-E.
The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 4 1235-1242
Copyright © 1997 by The Endocrine Society

Reproductive Endocrinology

Tocolytic Therapy with Fenoterol Induces Selective Down-Regulation of ß-Adrenergic Receptors in Human Myometrium1

Stefan Engelhardt, Wolfgang Zieger, Jan Kassubek, Martin C. Michel, Martin J. Lohse and Otto-Erich Brodde

Institut für Pharmakologie und Toxikologie der Universität Würzburg, Würzburg (S.E., M.J.L.); Frauenklinik (W.Z.), Mannheim; Medizinische Klinik und Poliklinik der Universität Essen (J.K., M.C.M., O.-E.B.), Essen; and Institut für Pharmakologie und Toxikologie der Universität Halle (O.-E.B.), Germany

Address all correspondence and requests for reprints to: O.-E. Brodde, Institut für Pharmakologie und Toxikologie, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Strasse 4, D-06097 Halle, Germany.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Tocolytic therapy with ß-adrenergic receptor agonists is a standard regimen to prevent preterm birth. Agonist exposure of ß-adrenergic receptors causes receptor desensitization in other organs, and this may limit the therapeutic value of ß-adrenergic receptor agonists. To study the effects of prolonged ß-adrenergic agonist treatment in human myometrium, we obtained biopsies during Caesarean section of 14 pregnant patients who had received fenoterol for at least 5 days and 14 untreated pregnant controls. The densities of total ß-adrenergic receptors, which are mainly of the ß2-subtype as assessed by [125I]iodo-cyanopindolol binding in crude membrane fractions, were more than 50% smaller in women receiving fenoterol, whereas {alpha}2-adrenergic receptor densities were similar. Gs and Gi G-protein {alpha}-subunit densities were unaltered as assessed by Western blotting and pertussis toxin-catalyzed [32P]ADP-ribosylation. ß-Adrenergic receptor kinase (ßARK) activity, as determined using bovine rhodopsin as the substrate, was the same in the two groups. Adenylyl cyclase activities in the presence of guanine nucleotides, NaF, forskolin, or Mn++ were also not altered by fenoterol treatment. The messenger RNA (mRNA) concentrations of ß2-adrenergic receptors, ßARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a reference), as determined by quantitative PCR, were unaffected by fenoterol treatment. We conclude that tocolysis with fenoterol results in a selective down-regulation of myometrial ß-adrenergic receptors, which is not associated with a reduction in the respective mRNA concentrations or alterations of {alpha}2-adrenergic receptors, Gs and Gi {alpha}-subunits, or ßARK activity or mRNA.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PRETERM labor is commonly treated with long-term application of ß-adrenergic receptor agonists to produce myometrial relaxation and prevent or delay preterm birth. However, the long-term efficacy and therapeutic usefulness of this approach have been repeatedly questioned (1). For example, the Canadian preterm investigators group (2) reported results of a large study indicating that treatment with the ß-adrenergic agonist, ritodrine, had no long-term benefits, produced no significant difference in the delay of delivery, and did not affect neonatal morbidity. Although this study has been criticized because of alleged underdosage of ritodrine (3, 4), and even though another large study reported beneficial effects of long-term tocolysis with iv terbutaline (5), these data raise the possibility that the usefulness of ß-adrenergic agonists in tocolysis may be limited.

Agonist-stimulation of ß-adrenergic receptors causes receptor desensitization in many cells and tissues (see Refs. 6 and 7 for reviews). Desensitization of agonist-stimulated ß-adrenergic receptors has also been observed in the myometrium. Lye et al. (8) induced preterm labor in sheep with mifepristone (RU486) and observed that iv ritodrine initially inhibited labor contractions but lost its effects within 16 h. This loss of efficacy was accompanied by a reduced cAMP-response to ß-adrenergic receptor stimulation and by reduced densities of ß-adrenergic receptors in the myometria. Other studies have also observed desensitization of myometrial ß-adrenergic receptors in sheep on prolonged agonist infusion (9, 10). In the human myometrium, ß-adrenergic receptor agonists have been shown to reduce the number of ß-adrenergic receptors, which were mainly of the ß2-subtype (11, 12). However, nothing is known about the underlying mechanisms leading to this decrease in receptor number in the myometrium.

ß-Adrenergic receptor desensitization can occur by multiple biochemical mechanisms. The two most important mechanisms are the rapid uncoupling between receptors and their G-proteins caused by phosphorylation of the receptors by the ß-adrenergic receptor kinases (ßARK) (13), followed by binding of the inhibitor protein ß-arrestin (14), and the much slower reduction of the receptor number (down-regulation) that evolves over many hours (15). When the different isoforms of ß-adrenergic receptors are expressed in identical cell lines, these desensitization mechanisms are most pronounced for the ß2-subtype, less pronounced for the ß1-subtype, and largely nonexistent for the ß3-subtype (16, 17).

The present study was undertaken to investigate the alterations that tocolysis might cause in the myometrial ß-adrenergic receptor system, including not only the receptors themselves, but also the ß-adrenergic receptor kinase and the stimulatory and inhibitory G-proteins as well as the Gi-protein coupled {alpha}2-adrenergic receptors. We report that a reduction of the ß2-adrenergic receptor number, without a concomitant reduction of the corresponding messenger RNA (mRNA), is the only alteration found in response to long-term tocolysis with fenoterol.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients and biopsy procurement

We studied myometrial biopsies obtained from 28 patients during Caesarean section after at least 5 days of fenoterol administration (n = 14) or from untreated control patients (n = 14). The studies had been approved by the ethical committee of the medical faculty, and all patients gave informed written consent. The clinical data of these patients are given in Table 1Go. All patients in the fenoterol group received fenoterol at the indicated doses, and all but one received MgSO4 to enhance tocolytic effects, and metoprolol to block ß1-adrenergic receptors. None of the patients from the control group received catecholamines or ß-adrenergic receptor antagonists. The average gestational age was 4 weeks higher in the control group because for ethical reasons tocolytic treatment was given to all women of low gestational age. Myometrial specimens of 0.5–3 g were removed from the free wall of the uterus at the time of the Caesarean section and placed in liquid nitrogen immediately after removal and stored at -80 C. Tissue procurement was the same for all biopsies. Unfortunately, the amount of available tissue did not allow to quantify each parameter in all patients.


View this table:
[in this window]
[in a new window]
  		Table 1. Clinical characteristics of patients undergoing Caesarean sections

 
Receptor binding studies

Crude membrane fractions were prepared from a fraction of the frozen tissue samples as previously described (12, 18). Briefly, a piece of frozen tissue was thawed and homogenized with an Ultra-Turrax (Janke & Kunkel, Staufen, Germany) in ice-cold buffer (0.25 M sucrose, 5 mM Tris-HCl, 1 mM EDTA, pH 7.4). The homogenate was centrifuged at 600 x g for 15 min, the supernatant filtered through four layers of medical gauze, and centrifuged at 50,000 x g for 20 min at 4 C. The resulting pellets were resuspended in 10 mM Tris-HCl, 154 mM NaCl buffer, p.H 7.4, that contained 0.55 mM ascorbic acid to yield a protein concentration of 0.5–1 mg/mL (for ß-adrenergic receptor assessment) or in 50 mM Tris-HCl, 0.5 mM EDTA buffer, pH 7.4, to yield a protein concentration of 2–3 mg/mL (for {alpha}2-adrenergic receptor assessment). The protein concentration was assessed by the method of Bradford (19) using bovine immunoglobulin G as standard.

ß2-Adrenergic receptors were quantitated by (-)[125I]-iodo-cyanopindolol (ICYP, SA 2200 Ci/mmol, New England Nuclear, Dreieich, Germany) binding with six concentrations ranging from 5–200 pM; nonspecific binding was defined as binding in the presence of 1 µM (±)-CGP 12177 (4-(3-tertiarybutylamino-2-hydroxypropoxyl-benzimidazole-2-on). The number of myometrial {alpha}2-adrenergic receptors was assessed by [3H]rauwolscine (SA 80 Ci/mmol, New England Nuclear) binding using six concentrations ranging from 0.5–10 nM; nonspecific binding was defined by the presence of 10 µM phentolamine.

Quantification of G-protein {alpha}-subunits

G-protein {alpha}-subunits were quantified by immunoblotting and pertussis toxin-catalyzed ADP-ribosylation as previously described in detail (20). Briefly, {alpha}-subunits of Gs and Gi were detected using the antisera RM/1 and AS/7 (New England Nuclear), respectively, at a 1:500 dilution, followed by quantitation on the blots with [125I]-protein A (SA 8.5 µCi/µg, New England Nuclear). Pertussis toxin-catalyzed ADP-ribosylation was performed by incubating crude myometrial membranes (50 µg) with activated pertussis toxin in the presence of [32P]-NAD (SA 30 Ci/mmol) for 60 min at 30 C. Proteins were resolved by SDS-PAGE, and the ADP-ribosylated bands of 39–41 kilodaltons were excised and quantitated by Cerenkov counting.

Determination of ßARK activity

Enzymatic activity of ßARK was measured as previously described (21, 22). Frozen myometrial tissue (100 mg) was homogenized for 30 sec with a polytron device in 1 mL 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and centrifuged for 10 min at 200,000 x g. The protein concentration in the supernatants was determined according to Bradford (19). Urea-treated rod outer segments containing >95% rhodopsin were prepared from bovine retinae as the substrate for ßARK. Aliquots (50 µg protein) of these cytosolic preparations were incubated with 500 pmol rhodopsin in the same buffer containing 10 mM MgCl2 and 0.3 mM [32P]ATP in a total volume of 60 µL. 32P incorporation into rhodopsin was determined by separation of the reaction mixture by SDS-PAGE and quantitation of the radioactivity in the rhodopsin band with a phosphoimager (Fuji, Tokyo, Japan).

Determination of mRNA concentrations

RNA from the frozen tissue samples was prepared by a shortened version of the protocol of Chomczinski and Sacchi (23) essentially as recently described (24, 25). The purity of the RNA was checked by measuring the ratio of the absorbance at 260 and 280 nm and was 1.8–2.0 in all cases. Aliquots (500 ng) of RNA were reverse transcribed into complementary DNA (cDNA) using random hexamers and Superscript II reverse transcriptase (GIBCO/BRL, Gaithersburg, MD) as described elsewhere (24). In all experiments, reactions containing no reverse transcriptase were done as negative controls.

Sense and antisense oligonucleotide primer pairs were synthesized to match the sequences of the human ß2-adrenergic receptor (26), human ßARK-I (21) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH (27). Details about the primers and the expected PCR products are given in Table 2Go. PCR was done with the transcript obtained from 50 ng RNA using 0.5 µM of the respective primers, 1.25 U Taq polymerase (Perkin Elmer, Norwalk, CT), 200 µM desoxynucleotides plus 0.3 µCi [{alpha}-32P]deoxycytidine triphosphate, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, in a volume of 50 µL. Amplifications were done in a Perkin-Elmer model 480 thermal cycler with denaturation at 94 C for 1 min (3 min in first cycle), annealing for 1 min at the temperatures indicated in Table 2Go, and an extension at 72 C for 1 min (10 min in last cycle). Multiple samples from different biopsies were assayed for each gene using a single master reaction mixture. The PCR products were isolated by electrophoresis on a 10% polyacrylamide gel. After drying on a gel dryer, the incorporated radioactivity was determined using a phosphoimager (Fuji).


View this table:
[in this window]
[in a new window]
  		Table 2. Sequences of forward and reverse oligonucleotide primers specific for human sequences of ß2-adrenergic receptor, ßARK, and GAPDH

 
In all experiments, a template-free negative control, RNA template not subjected to RT, and known amounts of plasmids containing the respective sequences were amplified to monitor the accuracy and efficiency of the PCR. Validation of this PCR technique in comparison with a solution hybridization assay was recently published from our laboratory (25); moreover, we have found a tight linear correlation between ß2-adrenergic receptor mRNA concentrations as assessed by this PCR method and Northern blotting in DDT1-MF2 smooth muscle cells (r = 0.997, n = 8, data not shown).

Adenylyl cyclase assays

Adenylyl cyclase activity was assessed as described by Salomon et al. (28) with minor modifications as detailed elsewhere (29). Two different incubation conditions were employed. For Gpp(NH)p, NaF, and forskolin activation, membranes (30–40 µg protein) were incubated for 10 min at 30 C in a final volume of 100 µL containing 40 mM HEPES buffer, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 500 µM [{alpha}-32P]ATP, 100 µM cAMP, and an ATP regenerating system (5 mM phosphocreatine and 50 U/mL creatine phosphokinase, buffer A). For Mn++ activation, membranes were incubated in buffer A without Mg++. The reaction was stopped by adding 0.8 mL 50 mM Tris-HCl buffer (pH 7.4 at 25 C) containing 40 mM ATP and 1.4 mM cAMP. [3H]cAMP (5,000–10,000 cpm) was then added to monitor the recovery of [32P]cAMP (28).

Statistical analysis

Data are expressed as mean ± SEM. Comparison between the two different groups was performed by two-tailed unpaired t tests. A P value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Down-regulation of ß2-adrenergic receptors by tocolysis

The high-affinity antagonist radioligand ICYP was used to quantitate ß-adrenergic receptors in human myometrial membranes, 85.5% of which are of the ß2-subtype (12). The number of binding sites for ICYP was 6.3 ± 0.6 fmol/mg protein in the control group but only 2.8 ± 0.4 fmol/mg protein in patients who had been treated with fenoterol (P < 0.01, Fig. 1AGo). On the other hand, ß-adrenergic receptor number in the control and in the tocolysis group was not significantly related to the gestational age (r2 0.2344 and 0.0497, respectively; P > 0.05 in both groups; Fig. 1BGo). The number of {alpha}2-adrenergic receptors was not significantly changed in the fenoterol treatment group (Fig. 1AGo).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. A, Effects of tocolysis with fenoterol on ß-adrenergic (left) and {alpha}2-adrenergic receptor density (right) in human myometrial membranes. Myometrial specimens of patients listed in Table 1Go were removed during Caesarian section, crude membranes were prepared, and ß- and {alpha}2-adrenergic receptor densities were determined as described in Subjects and Methods. Ordinates, left: myometrial ß-adrenergic receptor density in femtomoles (-)-[125I]ICYP specifically bound per milligrams protein, right: myometrial {alpha}2-adrenergic receptor density in femtomoles [3H]rauwolscine specifically bound per milligrams protein. Means ± SEM are given; number of experiments is shown at bottom of columns. Dissociation constant (KD) values for ICYP were: control, 7.6 ± 2.3 pM (n = 13); tocolysis, 11.0 ± 2.8 pM (n = 14); KD values for [3H]rauwolscine were: control, 0.72 ± 0.11 nM (n = 13); tocolysis, 0.89 ± 0.09 nM (n = 13). **, P < 0.01 vs. control. B, Relationship between ß-adrenergic receptor density in myometrial membranes and gestational age in fenoterol-treated ({blacksquare}) and nontreated pregnant women ({circ}). For details see Patients and Methods. Ordinate: myometrial ß-adrenergic receptor density in femtomoles ICYP specifically bound per milligrams protein; abscissa: gestational age of patients in weeks.

 
G-protein studies

Gs and Gi {alpha}-subunits were assayed by quantitative Western blotting using the RM/1 or AS/7 antiserum, respectively, in conjunction with [125I]-protein A detection. The densities of both classes of {alpha}-subunits were unaffected by fenoterol treatment (Table 3Go). Likewise, the concentration of substrates for pertussis toxin-catalyzed ADP-ribosylation, which corresponds to the {alpha}-subunits of the Go/Gi-family, were not different between the two groups of patients (Table 3Go). These results indicate that G-protein densities in human myometrium were not affected by tocolytic treatment.


View this table:
[in this window]
[in a new window]
  		Table 3. G-protein subunits in myometria of fenoterol-treated women and untreated controls

 
To test whether the functional activity of Gs and/or Gi might be altered, we assessed activation of myometrial adenylyl cyclase by Gpp(NH)p (acting at Gs and Gi), NaF [activating under these conditions predominantly Gs with little effects on Gi (30)], forskolin [acting predominantly at the catalytic unit of the adenylyl cyclase, but involving at least partly Gs (31)], and Mn++ [acting solely at the catalytic unit of the adenylyl cyclase (32)]. As shown in Fig. 2Go, Gpp(NH)p-, NaF-, forskolin-, and Mn++-induced adenylyl cyclase activation was not affected by fenoterol treatment, indicating that the functional activities of Gs, Gi, and the catalytic unit of adenylyl cyclase were not altered.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Effects of tocolysis with fenoterol on adenylyl cyclase activity in human myometrial membranes. For details see Patients and Methods. Ordinate: net increase in myometrial adenylyl cyclase activity upon stimulation in pmol cAMP formed/mg protein/min. Given are means ± SEM; number of experiments is in parentheses. B, Basal adenylyl cyclase activity; Gpp, Gpp(NH)p; FOR, forskolin.

 
ßARK activity

To test whether the ß-adrenergic receptor down-regulation is [as in heart failure (24, 33)], accompanied by increased ßARK expression, we measured the total ßARK activity in cytosolic preparations from the myometria. These assays were done using rhodopsin as the substrate, which can be phosphorylated not only by the predominant kinase, ßARK-I, but also by the other related members of this kinase family (13). Rhodopsin phosphorylation was readily obtained with cytosolic preparations from both control and fenoterol-treated patients (Fig. 3Go, inset). Quantitative analysis of these assays revealed almost identical activities in the two groups of patients (Fig. 3Go).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Effects of tocolysis with fenoterol on myometrial ßARK activity. Cytosolic preparations from myometrial specimens were assayed for their ability to phosphorylate ßARK-substrate rhodopsin in a light-dependent manner, and rhodopsin phosphorylation was visualized by SDS-PAGE and autoradiography (inset). Extent of phosphate incorporation was quantified with a phosphoimager. Data are mean ± SEM.

 
Quantification of mRNAs

To determine the steady state concentrations of the mRNAs for the ß2-adrenergic receptor and also for ßARK-I, quantitative RT-PCR were carried out with RNA prepared from human myometria. GAPDH, a housekeeping gene widely used for standardization in such experiments, was chosen as an endogenous control. The absolute values of the expression of GAPDH in these biopsies were very similar in both groups (data not shown). In agreement with our earlier data on myocardium (24, 25, 33), this indicates that this mRNA is not affected by ß-adrenergic stimulation and can therefore be used for normalization. Figure 4AGo shows the PCR products obtained for the ß2-adrenergic receptor, GAPDH, and ßARK-I. In all cases, a single well-defined product of the appropriate size was obtained, which could be used for quantification by phosphoimaging. Such quantifications are shown in Fig. 4BGo and demonstrate exponential amplification between cycles 30 and 34 for ßARK-I, cycles 21 and 25 for GAPDH, and cycles 28 and 34 for ß2-adrenergic receptor. Consequently, 32 cycles were chosen for the amplification of ßARK, 30 for the ß2-adrenergic receptor, and 23 for GAPDH. The efficiencies of amplification were almost identical, which allows the use of the GAPDH-product for normalization.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 4. PCR amplification of cDNAs for ß2-adrenergic receptor (ß2AR), GAPDH, and ßARK-I. PCRs were done with cDNA transcribed with random hexamers from 50 ng of total RNA. A, PCR products obtained after 30, 23, and 32 cycles, respectively. B, Amplification characteristics. After indicated number of cycles, 10-µL aliquots were taken from reaction mixture and subjected to polyacrylamide gel electrophoresis followed by autoradiography. [32P]Deoxycytidine triphosphate incorporation was used to quantify PCR products using a phosphoimager. Amplification efficiencies are given for the different PCR products. Data are presented as mean ± SEM from four different PCR reactions.

 
All biopsies were then screened simultaneously for the expression of the specific mRNAs. As expected from the activity measurements, the amounts of ßARK-I mRNA were the same in both groups (Fig. 5Go). Likewise, in spite of the marked reduction of the ß2-adrenergic receptor expression, fenoterol treatment had no effect on their mRNA (Fig. 5Go). This suggests that the down-regulation of the receptors occurs on the protein rather than the mRNA concentration.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Effects of tocolysis on mRNA concentrations of ßARK-I and ß2-adrenergic receptors (ß2AR) in myometria. PCRs of cDNA transcribed from 50 ng of total myometrial RNA were done and analyzed as described in Fig. 4Go. Values for ßARK-I and ß2-AR were normalized using corresponding values for GAPDH as a standard. Number of cycles was 23 for GAPDH, 30 for ß2-AR, and 32 for ßARK-I. Data are presented as mean ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Over the past years it has become clear that G-protein-coupled receptors are part of a complex and highly regulated signal transduction machinery. Although this appears to be important to maintain a physiological balance, such regulation may limit the therapeutic potential of receptor agonists as drugs. In fact, the effects of many receptor agonists vane over time. In the case of the ß-adrenergic receptors, the molecular mechanisms of this regulation have been studied in much detail in cultured cells (for recent reviews see Refs. 6 and 7). In addition, a large number of studies have addressed the regulation of these receptors in the human heart in chronic heart failure (34). Heart failure is characterized by enhanced sympathetic activity (35) leading to increased stimulation of these receptors, and this has been shown to cause a loss of responsiveness of the heart to the stimulatory effects of catecholamines (36). This loss of responsiveness has been traced back to reduced mRNA-concentrations of the ß1-adrenergic receptors (24, 37, 38), which represent the predominant subtype in the heart, and to increased concentrations of ßARK-I mRNA (33), which lead to increased concentrations of this kinase and thus presumably to uncoupling of receptors and G-proteins. Interestingly, however, mRNA concentrations of cardiac ß2-adrenergic receptors were unchanged in chronic heart failure (24, 37, 38), despite the fact that some studies have found a decrease in their number (for references see Ref.39). Another biochemical event that can contribute to a loss of ß-adrenergic receptor responsiveness is the up-regulation of inhibitory elements in the signal transduction cascade, i.e. Gi-protein or receptors coupled to it (cross-regulation, see Refs. 40 and 41). Thus, chronic activation of cardiac ß-adrenergic receptors is accompanied by an increase in the inhibitory G-protein Gi in human heart failure (42) and in rats following chronic infusion of isoprenaline (43, 44).

The present study was undertaken with the hypothesis that chronic activation of myometrial ß2-adrenergic receptors with ß2-adrenergic receptor agonists might cause similar changes as those observed for cardiac ß1-adrenergic receptors in chronic heart failure, and that the ensuing desensitization of the ß2-adrenergic receptor system might be a reason for the limited usefulness of this tocolytic therapeutic regimen. According to standard medical practice in Germany, it is not considered acceptable to withhold ß2-adrenergic agonist treatment in women in preterm labor. Moreover, a ß1-selective antagonist (e.g. metoprolol) and MgSO4 are added regularly to minimize concomitant stimulation of cardiac ß1-adrenoceptors and to enhance tocolytic efficacy, respectively. Therefore, the women receiving the ß2-adrenergic agonist differ from the control patients of our study in three additional ways, i.e. gestational age, metoprolol treatment, and MgSO4 treatment. Although we cannot rule out that any of these additional factors has affected the outcome of our study, we feel that the fenoterol treatment had the dominant effect for the following reasons: to our knowledge MgSO4 has never been shown to affect ß-adrenergic receptor function or desensitization, and no mechanism to this effect is known. Although treatment with ß1-selective antagonists such as metoprolol can up-regulate ß1-adrenergic receptors, the density of ß2-adrenergic receptors was not affected in such studies (45). A more serious problem is whether different gestational ages might have contributed to the observed data in our study. For example, it has been observed that human myometrial ß-adrenergic receptors undergo functional uncoupling towards the end of pregnancy, i.e. in gestational weeks 39–40 (46). However, we feel that this cannot explain the reduction of ß-adrenergic receptors seen in our study for several reasons. First, gestational age and myometrial ß-adrenergic receptor density were not significantly correlated within the control or tocolysis group. Second, ß-adrenergic receptor density was also lower in the tocolysis group in the subset of patients with similar gestational ages in both groups (cf. Fig. 1BGo). Third, the data by Litime et al. (46) imply functional uncoupling towards the end of pregnancy, which, if anything, would result in an understimation of the desensitization in tocolysis patients with a smaller gestational age compared with control term women. Therefore, it appears that the ß2-adrenergic agonist treatment indeed is the most likely cause of our observations. Confirmation in a study design with random allocation of tocolytic treatment would be useful, but that is not considered ethically acceptable in Germany.

The overall pattern of changes in the present study was quite different from that observed for ß1-adrenergic receptors in heart failure: we found that tocolysis with fenoterol caused an isolated decrease of receptor densities, without a concomitant decrease of the corresponding mRNA. Neither the activity or mRNA concentration of ßARK-I nor the concentrations or functional activities of the relevant G-protein {alpha}-subunits were altered. Furthermore, we found no evidence for a cross-regulation of {alpha}2-adrenergic receptors.

A possible reason for this discrepancy might lie in the fact that whereas heart failure develops over very long periods of time, tocolysis in our patients was only performed for days, and that the other changes observed in heart failure might appear only later. However, application of ß-adrenergic receptor agonists to rats for 4 days has been reported to cause not only down-regulation of cardiac ß-adrenergic receptors, but also receptor uncoupling as well as up-regulation of Gi {alpha}-subunits (43, 44). Furthermore, rapidly evolving experimental models of heart failure in dogs have been reported to decrease ß-adrenergic receptor number und coupling, Gs {alpha}-subunit expression, and adenylyl cyclase (for references see Ref.34). Thus, the cardiac ß-adrenergic receptor system of several species reacts to changes in ß-adrenergic stimulation with an array of changes in a matter of days. This suggests that the different patterns of changes in the cardiac and myometrial ß-adrenergic receptor systems are not caused by the relatively short duration of ß-adrenergic receptor activation during tocolysis. On the other hand, similar to the myometrium (present study), we recently found that a 2-week treatment of healthy volunteers with the ß2-adrenergic receptor agonist terbutaline markedly reduced lymphocyte ß2-adrenergic receptor number but did not affect lymphocyte Gi-densities (47), and that treatment of the human neuroblastoma cell line, SK-N-MC, with isoprenaline for 24 h caused a marked decrease in ß1-adrenergic receptor number but did not change Gi-densities (48); extending the incubation to 4 days also did not lead to any changes in Gi (our unpublished observations).

A second possible explanation for these differences is that whereas the cardiac ß-adrenergic receptors are mostly of the ß1-subtype (39), the ß2-subtype predominates in the myometrium (11, 12). However, when expressed in the same cell line, the ß2-subtype has been shown to be more readily regulated than the ß1-subtype (16). Furthermore, cross-regulation between ß-adrenergic receptors and Gi {alpha}-subunits and {alpha}2-adrenergic receptors has been demonstrated much better for the ß2- than for the ß1-subtype (49, 50). And finally, alterations in Gi {alpha}-subunit expression are thought to be cAMP-mediated (42), and both the ß1- and the ß2-subtype are coupled to increases in cAMP. Thus, there is no reason to assume that in a tissue expressing ß2-adrenergic receptors the regulatory response to ß-receptor agonists would be more limited than in a tissue expressing the ß1-subtype.

Taken together, the most reasonable explanation for the different types of regulation of myometrial and myocardial ß-adrenergic receptors is the hypothesis that there are tissue or organ-specific factors that determine the pattern of receptor desensitization. Such tissue-specific factors might be the expression of specific regulatory proteins, such as the receptor kinases, phosducins, or mRNA-regulatory proteins (51, 52, 53).

It is interesting to note that tocolysis causes a reduction of myometrial ß2-adrenergic receptor densities without a concomitant reduction of the corresponding mRNA. In many studies on isolated cells, a similar time course for the agonist-induced down-regulation of ß2-adrenergic receptors and their mRNA has been observed, and this mRNA reduction has been suggested to be an essential mechanism of receptor down-regulation (54, 55, 56, 57). This may indeed be true for cell culture lines, because a continuous synthesis is necessary to maintain receptor densities in a rapidly dividing cell population. However, in the tissues of intact animals, ß-adrenergic receptors have a half-life of many days to several weeks (58). Thus, reduced mRNA concentrations should not be able to induce substantial down-regulation of the receptors themselves in a matter of a few days (7), and in fact, we did not even find any reduction in myometrial ß2-adrenergic receptor mRNA after tocolysis. The down-regulation of these receptors by tocolysis must therefore be caused by degradation of the receptors themselves.

In summary, our study indicates that tocolytic treatment with fenoterol caused only a single change in the myometrial ß2-adrenergic receptor system: an isolated down-regulation of the receptors that was most likely caused by direct degradation of the receptors. Comparison of these changes with those observed in the cardiac ß-adrenergic receptor systems underlines the importance of tissue specific factors in receptor regulation. It appears likely that the down-regulation of myometrial ß-adrenergic receptors limits the usefulness of ß-adrenergic receptor agonists as tocolytic agents.


   Footnotes
 
1 This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (SFB 355 to M.J.L., Br 526/3–2 to O.-E.B., Ph 41/1–3 to M.C.M.), and the Fonds der Chemischen Industrie (to M.J.L.). Back

Received June 12, 1996.

Revised November 6, 1996.

Accepted January 1, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Leveno KJ, Cunningham FG. 1992 ß-Adrenergic agonists for preterm labor. New Engl J Med. 327:349–351.[Medline]
  2. The Canadian Preterm Labor Investigators Group. 1992 Treatment of preterm labor with the beta-adrenergic agonist ritodrine. New Engl J Med. 327:308–312.[Abstract]
  3. Caritis SN. 1992 Treatment of preterm labor with the beta-adrenergic agonist ritodrine. New Engl J Med. 327:1758.[Medline]
  4. Schiff E, Sivan E, Terry S, et al. 1993 Currently recommended oral regimens for ritodrine tocolysis result in extremely low plasma levels. Am J Obstet Gynecol. 169:1059–1064.[Medline]
  5. Kosasa TS, Busse R, Wahl N, Hirata G, Nakayama RT, Hale RW. 1994 Long-term tocolysis with combined intravenous terbutaline and magnesium sulfate: a 10-year study of 1000 patients. Obstet Gynecol. 84:369–373.[Abstract/Free Full Text]
  6. Hausdorff WP, Caron MG, Lefkowitz RJ. 1990 Turning off the signal: desensitization of ß-adrenergic receptor function. FASEB J. 4:2881–2889.[Abstract]
  7. Lohse MJ. 1993 Molecular mechanisms of membrane-receptor desensitization. Biochim Biophys Acta. 1179:171–188.[Medline]
  8. Lye SJ, Dayes BA, Freitag CL, Brooks J, Casper RF. 1992 Failure of ritodrine to prevent preterm labor in sheep. Am J Obstet Gynecol. 167:1399–1408.[Medline]
  9. Caritis SN, Chiao JP, Moore JJ, Ward SM. 1987 Myometrial desensitization after ritodrine infusion. Am J Physiol. 253:E410–E417.
  10. Caritis SN, Chiao JP, Kridgen P. 1991 Comparison of pulsatile and continuous ritodrine administration: effects on uterine contractility and ß-adrenergic receptor cascade. Am J Obstet Gynecol. 164:1005–1012.[Medline]
  11. Berg G, Andersson RGG, Ryden G. 1985 ß-Adrenergic receptors in human myometrium during pregnancy: changes in the number of receptors after ß-mimetic treatment. Am J Obstet Gynecol. 151:392–396.[Medline]
  12. Michel MC, Pingsmann A, Nohlen M, Siekmann U, Brodde O-E. 1989 Decreased myometrial beta-adrenoceptors in women receiving beta-2-adrenergic tocolytic therapy: correlation with lymphocyte beta-adrenoceptors. Clin Pharmacol Ther. 45:1–8.[Medline]
  13. Premont RT, Inglese J, Lefkowitz RJ. 1995 Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J. 9:175–182.[Abstract]
  14. Lohse MJ, Benovic JL, Codina J, Caron MG, Lefkowitz RJ. 1990 ß-Arrestin: a protein that regulates ß-adrenergic receptor function. Science. 248:1547–1550.[Abstract/Free Full Text]
  15. Collins S, Caron MG, Lefkowitz RJ. 1991 Regulation of adrenergic receptor responsiveness through modulation of receptor gene expression. Annu Rev Physiol. 53:497–508.[CrossRef][Medline]
  16. Suzuki T, Nguyen CT, Nantel F, et al. 1992 Distinct regulation of ß1- and ß2-adrenergic receptors in Chinese hamster fibroblasts. Mol Pharmacol. 41:542–548.[Abstract]
  17. Nantel F, Bonin H, Emorine LJ, et al. 1993 The human ß3-adrenergic receptor is resistant to short term agonist-promoted desensitization. Mol Pharmacol. 43:548–555.[Abstract]
  18. Brodde O-E, Seher U, Nohlen M, Fischer MW, Michel MC. 1988 Correlation between myometrial and platelet {alpha}2-adrenoceptor density. Eur J Pharmacol. 150:403–404.[CrossRef][Medline]
  19. Bradford MM. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72:248–254.[CrossRef][Medline]
  20. Michel-Reher M, Gross G, Jasper JR, et al. 1993 Tissue- and subunit-specific regulation of G-protein expression by hypo- and hyperthyroidism. Biochem Pharmacol. 45:1417–1423.[CrossRef][Medline]
  21. Benovic JL, Mayor F, Staniszewski C, Lefkowitz RJ, Caron MG. 1987 Purification and characterization of the ß-adrenergic receptor kinase. J Biol Chem. 262:9026–9032.[Abstract/Free Full Text]
  22. Söhlemann P, Hekman M, Buchen C, Elce JS, Lohse MJ. 1993 Purification and functional characterization of ß-adrenergic receptor kinase expressed in insect cells. FEBS Lett. 324:59–62.[CrossRef][Medline]
  23. Chomczinski P, Sacchi N. 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-chloroform extraction. Anal Biochem. 162:156–159.[Medline]
  24. Ungerer M, Böhm M, Elce JS, Erdmann E, Lohse MJ. 1993 Altered expression of beta-adrenergic-receptor-kinase and beta-1-adrenergic receptors in the failing human heart. Circulation. 87:454–463.[Abstract/Free Full Text]
  25. Engelhardt S, Böhm M, Erdmann E, Lohse MJ. 1996 Analysis of beta-adrenergic receptor mRNA levels in human ventricular biopsy specimens by quantitative polymerase chain reactions: progressive reduction of beta1-adrenergic mRNA in heart failure. J Am Coll Cardiol. 27:146–154.[Abstract]
  26. Kobilka BK, Dixon RAF, Frielle T, et al. 1987 cDNA for the human ß2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc Natl Acad Sci USA. 84:46–50.[Abstract/Free Full Text]
  27. Hanauer A, Mendel JL. 1984 The glyceraldehyde 3 phosphate dehydrogenase gene family: structure of human cDNA and of an x chromosome linked pseudogene; amazing complexity of the gene family in the mouse. EMBO J. 3:2627–2633.[Medline]
  28. Salomon Y, Londos C, Rodbell M. 1974 A highly sensitive adenylate cyclase assay. Anal Biochem. 58:541–548.[CrossRef][Medline]
  29. Brown L, Deighton NM, Bals S, et al. 1992 Spare receptors for ß-adrenoceptor-mediated positive inotropic effects of catecholamines in the human heart. J Cardiovasc Pharmacol. 19:222–232.[Medline]
  30. Katada T, Northup JK, Bokoch GM, Ui M, Gilman AG. 1984 The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and guanine nucleotide-dependent hormonal inhibition. J Biol Chem. 259:3578–3585.[Abstract/Free Full Text]
  31. Darfler FJ, Mahan LC, Koachman AM, Insel PA. 1985 Stimulation by forskolin of intact S49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase. J Biol Chem. 257:11901–11907.[Free Full Text]
  32. Strittmatter S, Neer EJ. 1980 Properties of the separated catalytic and regulatory units of brain adenylate cyclase. Proc Natl Acad Sci USA. 77:6344–6348.[Abstract/Free Full Text]
  33. Ungerer M, Parruti G, Böhm M, et al. 1994 Expression of ß-arrestins and ß-adrenergic receptor kinases in the failing human heart. Circ Res. 74:206–213.[Abstract/Free Full Text]
  34. Brodde O-E, Michel MC, Zerkowski H-R. 1995 Signal transduction mechanisms controlling cardiac contractility and their alterations in chronic heart failure. Cardiovasc Res. 30:570–584.[CrossRef][Medline]
  35. Cohn JN. 1990 Abnormalities of peripheral sympathetic nervous system control in congestive heart failure. Circulation [Suppl I]. 82:59–67.
  36. Bristow MR, Ginsburg R, Minobe W, et al. 1982 Decreased catecholamine sensitivity and ß-adrenergic-receptor density in failing human hearts. New Engl J Med. 307:205–211.[Abstract]
  37. Bristow MR, Minobe WA, Raynolds MV, et al. 1993 Reduced ß1 receptor messenger RNA abundance in the failing human heart. J Clin Invest. 92:2737–2745.
  38. Ihl-Vahl R, Eschenhagen T, Kübler W, et al. 1996 Differential regulation of mRNA specific for ß1- and ß2-adrenergic receptors in human failing hearts. Evaluation of the absolute cardiac mRNA levels by two independent methods. J Mol Cell Cardiol. 28:1–10.[CrossRef][Medline]
  39. Brodde O-E. 1991 ß1- and ß2-adrenoceptors in the human heart: properties, function, and alterations in chronic heart failure. Pharmacol Rev. 43:203–241.[Medline]
  40. Milligan G, Green A. 1991 Agonist control of G-protein levels. Trends Pharmacol Sci. 12:207–209.[CrossRef][Medline]
  41. Reithmann C, Gierschik P, Werdan K, Jakobs KH. 1991 Role of inhibitory G protein {alpha}-subunits in adenylyl cyclase desensitization. Mol Cell Endocrinol. 82:C215–C221.
  42. Eschenhagen T. 1993 G proteins and the heart. Cell Biol Int. 17:723–749.[CrossRef][Medline]
  43. Mende U, Eschenhagen T, Geertz B, et al. 1992 Isoprenaline-induced increase in the 40/41 kDa pertussis toxin substrates and functional consequences on contractile response in rat heart. Naunyn-Schmiedeberg’s Arch Pharmacol. 345:44–50.[Medline]
  44. Eschenhagen T, Mende U, Diederich M, et al. 1992 Long term ß-adrenoceptor-mediated up-regulation of Gi{alpha} and Go{alpha} mRNA levels and pertussis toxin-sensitive guanine nucleotide-binding proteins in rat heart. Mol Pharmacol. 42:773–783.[Abstract]
  45. Michel MC, Pingsmann A, Beckeringh JJ, Zerkowski H-R, Doetsch N, Brodde O-E. 1988 Selective regulation of ß1- and ß2-adrenoceptors in the human heart by chronic ß-adrenoceptor antagonist treatment. Br J Pharmacol. 94:685–692.[Medline]
  46. Litime M-H, Pointis G, Breuller M, Cabrol D, Ferre F. 1989 Disappearance of ß-adrenergic response of human myometrial adenylate cyclase at the end of pregnancy. J Clin Endocrinol Metab. 68:1–6.
  47. Brodde O-E, Michel-Reher M, Oefler D, Sarikouch S, Michel MC. 1992 Terbutaline-induced downregulation of ß2-adrenoceptors without Gi-protein alterations in human lymphocytes. J Cardiovasc Pharmacol. 20:785–789.[Medline]
  48. Michel MC, Feth F, Sundermann M, Rascher W, Brodde O-E. 1993 ß-Adrenoceptor desensitization in SK-N-MC cells, a human cell line containing a homogeneous population of ß1-adrenoceptors. J Auton Pharmacol. 13:425–438.[CrossRef]
  49. Hadcock JR, Ross M, Watkins DC, Malbon CC. 1990 Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein Gi{alpha}2. J Biol Chem. 265:14784–14790.[Abstract/Free Full Text]
  50. Morris GM, Hadcock JR, Malbon CC. 1991 Cross-regulation between G-protein-coupled receptors. Activation of ß2-adrenergic receptors increases {alpha}1-adrenergic receptor mRNA levels. J Biol Chem. 266:2233–2238.[Abstract/Free Full Text]
  51. Bauer PH, Müller S, Puzicha M, et al. 1992 Phosducin is a protein kinase A-regulated G-protein regulator. Nature. 358:73–76.[CrossRef][Medline]
  52. Schröder S, Lohse MJ. 1996 Inhibition of G-protein ß{gamma}-subunit functions by phosducin-like protein. Proc Natl Acad Sci USA. 93:2100–2104.[Abstract/Free Full Text]
  53. Port JD, Huang L-Y, Malbon CC. 1992 ß-Adrenergic agonists that down-regulate receptor mRNA up-regulate a Mr 35,000 protein(s) that selectively binds to ß-adrenergic receptor mRNAs. J Biol Chem. 267:24103–24108.[Abstract/Free Full Text]
  54. Hadcock JR, Malbon CC. 1988 Down-regulation of ß-adrenergic receptors: agonist-induced reduction in receptor mRNA levels. Proc Natl Acad Sci USA. 85:5021–5025.[Abstract/Free Full Text]
  55. Hadcock JR, Malbon CC. 1988 Regulation of ß-adrenergic receptors by "permissive" hormones: glucocorticoids increase steady-state levels of receptor mRNA. Proc Natl Acad Sci USA. 85:8415–8419.[Abstract/Free Full Text]
  56. Hadcock JR, Malbon CC. 1989 Agonist regulation of ß-adrenergic receptor mRNA. Analysis in S49 mouse lymphoma mutants. J Biol Chem. 264:13956–13961.[Abstract/Free Full Text]
  57. Bouvier M, Collins S, O’Dowd BF, et al. 1989 Two distinct pathways for cAMP-mediated down-regulation of the ß2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level. J Biol Chem. 264:16786–16792.[Abstract/Free Full Text]
  58. Mahan LC, McKernan RM, Insel PA. 1987 Metabolism of alpha- and beta-adrenergic receptors in vitro and in vivo. Annu Rev Pharmacol Toxicol. 27:215–235.[Medline]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
T. Croci, R. Cecchi, P. Marini, C. Rouget, N. Viviani, G. Germain, F. Guagnini, Y. Fradin, L. Descamps, M. Pascal, et al.
In Vitro and in Vivo Pharmacological Characterization of Ethyl-4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl)amino]-phenoxy}propyl) Amino]cyclohexyl}benzoate Hydrochloride (SAR150640), a New Potent and Selective Human beta3-Adrenoceptor Agonist for the Treatment of Preterm Labor
J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 1118 - 1126.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
C. Rouget, O. Barthez, F. Goirand, M.J. Leroy, M. Breuiller-Fouche, Z. Rakotoniaina, P. Guerard, E.J. Morcillo, C. Advenier, P. Sagot, et al.
Stimulation of the ADRB3 Adrenergic Receptor Induces Relaxation of Human Placental Arteries: Influence of Preeclampsia
Biol Reprod, January 1, 2006; 74(1): 209 - 216.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
T. Frambach, T. Muller, S. Freund, S. Engelhardt, M. Sutterlin, M. J. Lohse, and J. Dietl
Self-Limitation of Intravenous Tocolysis with {beta}2-Adrenergic Agonists Is Mediated through Receptor G Protein Uncoupling
J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2882 - 2887.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
C. Rouget, M. Bardou, M. Breuiller-Fouche, C. Loustalot, H. Qi, E. Naline, T. Croci, D. Cabrol, C. Advenier, and M. J. Leroy
{beta}3-Adrenoceptor Is the Predominant {beta}-Adrenoceptor Subtype in Human Myometrium and Its Expression Is Up-Regulated in Pregnancy
J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1644 - 1650.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
T. Teli, D. Markovic, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos
Regulation of Corticotropin-Releasing Hormone Receptor Type 1{alpha} Signaling: Structural Determinants for G Protein-Coupled Receptor Kinase-Mediated Phosphorylation and Agonist-Mediated Desensitization
Mol. Endocrinol., February 1, 2005; 19(2): 474 - 490.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
S. GSELL, T. ESCHENHAGEN, G. KASPAREIT, M. NOSE, H. SCHOLZ, O. BEHRENS, and T. WIELAND
Apparent up-regulation of stimulatory G-protein {alpha} subunits in the pregnant human myometrium is mimicked by elevated smoothelin expression,
FASEB J, January 1, 2000; 14(1): 17 - 26.
[Abstract] [Full Text]

Home page
 Pharmacol. Rev.Home page
O.-E. Brodde and M. C. Michel
Adrenergic and Muscarinic Receptors in the Human Heart
Pharmacol. Rev., December 1, 1999; 51(4): 651 - 690.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J.-L. Lécrivain, J. Cohen-Tannoudji, M.-T. Robin, N. Coudouel, C. Legrand, and J.-P. Maltier
Molecular Mechanisms of Adenylyl Cyclase Desensitization in Pregnant Rat Myometrium following In Vivo Administration of the ss-Adrenergic Agonist, Isoproterenol
Biol Reprod, July 1, 1998; 59(1): 45 - 52.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Engelhardt, S.
Right arrow Articles by Brodde, O.-E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow