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â2-adrenoceptor desensitization in non-pregnant estrogen-primed rat myometrium involves modulation of oxytocin receptor gene expression
T Engstrøm1,2, P Bratholm1, H Vilhardt2 and N J Christensen1 1Department of Internal Medicine and Endocrinology, Herlev Hospital, University of Copenhagen, Denmark 2Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark (Requests for offprints should be addressed to T Engstrøm, Department of Endocrinology, Herlev Hospital, University of Copenhagen, 2730 Herlev, Denmark)
ABSTRACT The nona-peptide oxytocin (OT) induces con- mRNA levels or isoproterenol-induced relaxation of traction of the myometrium by interaction with isolated uterine strips. specific plasma membrane associated OT receptors Treatment with OT had no effect on the amount (OTR), whereas stimulation of â2-adrenoceptors of myometrial OTR mRNA. We have previously (â2AR) causes relaxation. Homologous desensitiz- found that OT down-regulates OTR in the ation of the myometrium to both hormones has non-pregnant rat myometrium, but this therefore been described. However, a possible inter- does not appear to take place at the level of mRNA action between the two systems has not been production. investigated. Isoproterenol treatment resulted in a three-fold In the present study, long-term in vivo treatment increase in OTR mRNA. This was accompanied by of non-pregnant estrogen-primed rats with iso- a 91% rise in OTR binding and an augmented proterenol decreased maximal relaxation of isolated contractile response of isolated uterine strips to OT, uterine strips challenged with isoproterenol. suggesting that the increased production of mRNA Increased EC50 values of similarly treated animals reflects formation of active receptors. Neither OTR suggest that the coupling between receptor occu- affinity nor EC50 of in vitro strips was affected by pancy and contractile response was impaired. isoproterenol treatment. We conclude that stimula- Since â2AR mRNA levels were left unchanged, tion of â2AR causes heterologous up-regulation of we conclude that the homologous desensitization OTR in the non-pregnant estrogen-primed rat to â2 stimulation is not due to changes in â2AR myometrium. gene expression. OT infusion did not alter â2AR Journal of Molecular Endocrinology (1998) 20, 261–270
INTRODUCTION described a cAMP-independent kinase (âAR kinase) which specifically phosphorylates â2ARs in lym- â-adrenomimetics are well known as potent inhibi- phoma cells when occupied by agonists. Lohse et al. tors of myometrial contractions (Anderson et al. (1989) found this kinase to be essential in 1975, Berg et al. 1982, Caritis et al. 1983) and are phosphorylation of âARs in human epidermoid used clinically in the treatment of pre-term labour. carcinoma cells. Upon phosphorylation âARs are However, continued occupancy of â2-adrenoceptors uncoupled from stimulatory G-proteins (Benovic (â2AR) leads to loss of myometrial responsiveness to et al. 1988) followed by internalization and subsequent agonist stimulation. This process, which degradation (Fishman & Perkins 1988). Using is known as homologous desensitization, involves receptor-binding studies, Dayes & Lye (1990) several steps. In frog erythrocytes (Sibley et al. observed down-regulation of âARs in ovine myo- 1985) and mouse lymphoma cells (Strasser et al. metrium challenged with isoproterenol, suggesting 1986) âAR desensitization is accompanied by either receptor internalization or reduced de novo receptor phosphorylation. Benovic et al. (1986) have receptor synthesis.
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We recently reported that pregnancy in itself prior to decapitation the rats received an i.m. induces desensitization of rat myometrium to injection of 75 µg estradiol benzoate. isoproterenol and that this primarily occurs at the level of gene expression (Engstrøm et al. 1997). Yeagley et al. (1987) found the degree of rat Primers and construction of internal mRNA myometrial tachyphylaxis to isoproterenol to standard increase with advanced gestational age. These The oligonucleotide primers (DNA Technology, findings suggest that other factors besides âAR Aarhus, Denmark) used for detection of â2AR stimulation are involved in the regulation of mRNA were the same as described for rat collecting myometrial â-adrenergic desensitization in duct (Mandon et al. 1995): sense primer 5*-TCT pregnancy. TCG AAA ACC TAT GGG AAC GGC-3* Engstrøm et al. (1988a) showed that in vivo (nucleotide 1036–1059), and antisense primer 5*- infusion of the oxytocin (OT) antagonist atosiban GGA TGT GCC CCT TCT GCA AAA TCT-3* led to up-regulation of OT receptors (OTR) in the (nucleotide 1355–1378). non-pregnant estrogen-dominated myometrium. In The primers used for OTR mRNA detection delivering rats â2AR mRNA increased compared were: sense primer 5*-GGG ACG TCA ATG CGC with day 21 of pregnancy (Engstrøm T, Bratholm P, CCA AGG AA-3* (nucleotide 2816–2838), and Vilhardt H & Christensen NJ, unpublished data). antisense primer 5*-ACC AAT AGA CAC CTA This increase was associated with a dramatic up- ATG CA-3* (nucleotide 3921–3940). regulation of OTR, suggesting that the imposition A basic local alignment search tool (Altschul et al. of a relaxing stimulus on the pregnant myometrium 1990) was used to search all non-redundant might induce formation of OTR. Hinko & Soloff databases (GenBank+EMBL+DDBJ+PDB) for se- (1993) induced OTR formation in rabbit term quence homology. No homology with any known amnion by raising cAMP levels with forskolin. products other than the actual receptors was found. In the present study we have measured â2AR The amplified DNA fragments consisted of mRNA in estrogen-dominated rat myometrium 342 bp (â2AR) and 375 bp (OTR). The exact following in vivo infusion of isoproterenol. To identities of the PCR products were confirmed by investigate whether â2-desensitization involves sequencing. The relevant peaks were collected from heterologous up-regulation of OTR we have the HPLC effluent and 1/100 volume 30% acetic concomitantly measured OTR mRNA. The physio- acid was added to adjust pH. Following addition logical relevance of receptor mRNA levels was of 50 µg glycogen the PCR products were precipi- evaluated by measurements of the relaxing and tated with an equal volume of isopropanol and contracting effects of isoproterenol and OT were finally washed with 70% ethanol and dried. respectively on isolated uterine strips. Sequencing was performed by the Applied Bio- systems dRhodamin sequencing kit (Perkin Elmer, Allerød, Denmark) using Applied Biosystems ABI MATERIALS AND METHODS 310 apparatus for separation and fluorescence detection. Animals Using a Polymerase Chain Reaction-MIMIC Female Wistar rats (250–350 g) were maintained construction kit (Clontech, Palo Alto, CA, USA) an under controlled conditions in the Panum Institute internal 240 bp DNA standard was constructed Animal House. Food and water were freely (Engstrøm et al. 1997). available. All experiments conformed to the The internal standard RNAs were constructed Guidelines on the Handling and Training of mainly as described by Faure et al. (1995). A Laboratory Animals (UFAW). composite primer, comprising 37 nucleotides of Rats were anaesthetized with a mixture of bacteriophage T7 RNA-polymerase promoter fol- Dormicum (1·25 mg, Hoffmann-La-Roche, Basel, lowed by the sequences of our usual sense primers, Switzerland) and Hypnorm (0·4 ml, Janssen was used for amplification of the 240 bp DNA Chimica, Geel, Belgium). Subsequently an incision sequence by PCR. The resulting 277 bp product in the lower abdominal wall was made and an was re-amplified using our antisense primers and a osmotic mini-pump (Alzet, Palo Alto, CA, USA) primer consisting of the initial 23 nucleotides of the was introduced into the abdominal cavity. The T7 RNA-polymerase promoter region. Following pumps had pumping rates of 1 µl/h and were filled HPLC purification the re-amplified products were with OT (0·5 mg/ml, Fehrring Pharmaceuticals, used for production of RNA by in vitro transcrip- Malmo¨, Sweden) or isoproterenol (10–20 mg/ml, tion (Riboprobe, Promega, Madison, WI, USA). Sigma Chem. Co., St Louis, MO, USA). Two days The resulting RNA standards were quantitated by
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UV detection at 260 nm (Gene-quant, Pharmacia, (Amersham International, Hillerød, Denmark; Stockholm, Sweden). Subsequently the RNA specific activity 35·0 Ci/mmol). The assay was standards underwent reverse transcription (RT) in performed at room temperature and initiated by the order to verify that the resulting products were addition of plasma membranes. At the end of the indistinguishable from the internal DNA standard. incubation period bound ligand was separated from unbound by filtration. Specific binding of [3H]OT was calculated by subtraction of binding in the RT-PCR presence of a 1000-fold excess of unlabeled OT and Polyadenylated mRNA from homogenized rat was finally related to protein content determined myometrium was isolated using the PolyATract according to Lowry et al. (1951). system 1000 (Promega) (Engstrøm et al. 1997). RT was performed in a mixture consisting of 250 µM dNTP, 40 U MMLV-RT (Promega), 31·2 U RNA- In vitro examination of contractile force of guard, 200 pmol antisense primer and 5 µl internal myometrial strips RNA standard in Promega RT-buffer. Incubations In vitro contractility was measured basically as were carried out for 60 min at 37 )C and the described previously (Engstrøm et al. 1997). One resulting cDNA used immediately or stored at uterine horn was opened longitudinally and a "80 )C. middle segment measuring 5 mm was mounted in PCR was carried out with 5 µl cDNA, 37·5 µM of an isometric myograph placed in an organ bath each dNTP, 1·0 U Taq polymerase (Pharmacia, containing Krebs–Ringer buffer. The tissue was Sweden), 40 pmol of both sense primer and anti- pre-contracted with 50 mM KCl and the relaxing sense primer in PCR buffer (10 mM Tris–HCl, effect of isoproterenol over a range of 10"10 to "6 50 mM KCl, 1·5 mM MgCl2, pH 9·0). Amplifica- 4#10 M was examined. The addition of iso- tion took place in a Perkin Elmer Model 460 proterenol was done in a cumulative manner to thermocycler. Cycling parameters were: 95 )C for obtain increasing concentrations and phentolamine 2 min followed by 27 cycles consisting of 1·5 min at (Regitin, Ciba-Geigy, Basel, Switzerland) at a 94 )C, 60 )C for 2 min and 72 )C for 2 min (â2AR) concentration of 1 µg/ml was present in the in- or 27 cycles consisting of 45 s at 94 )C, 56 )C for 45 s cubation medium to block adrenergic á-receptors. and 70 )C for 2 min (OTR). After the last cycle the The effect of each dose of isoproterenol was allowed incubations continued for 5 min at 72 )C followed by to level off before addition of the next. It was lowering of the temperature to 4 )C. PCR products ensured that the contractile response to KCl in itself were used immediately or stored at "80 )C. did not significantly diminish during the period of Quantitation of PCR products was carried out by the experiment. To ensure that the relaxing effect of means of HPLC using a TSK DEAE–NPR column isoproterenol was not affected by OT potentially (Engstrøm et al. 1997). Following chromatography, liberated from the endometrium, experiments in PCR products were UV detected at 254 nm. The which OTR were blocked with atosiban in the organ areas of the 240 bp PCR products of the internal chamber were also performed. standards represented 0·024 amol (â2AR) and Contractile responsiveness to OT doses in the 0·050 amol (OTR) respectively. Hence the amounts range of 0·63#10"10 to 0·13#10"6 M was of the 342 bp and the 375 bp PCR products could measured on isolated strips mounted in Munsick’s be quantitated relative to these standards and they buffer, pH 7·4 (Munsick 1960). Responses following were finally related to tissue wet weight. The isoproterenol or OT stimulation were expressed as a average value of the uterine horns of each animal percentage of the potassium-induced contraction was calculated to represent the amount of specific and were plotted against the logarithm of the mRNA. agonist concentration.
Binding of [3H]oxytocin to isolated Data analysis myometrial plasma membranes A computer program (Fig.P., Biosoft, Cambridge, Preparation of rat myometrial plasma membranes UK) was used for analysis of binding assays and was performed by subcellular fractionation contractile experiments. From curve fitting of (Engstrøm et al. 1988b). Subsequent binding of myometrial responsiveness to isoproterenol and OT [3H]oxytocin ([3H]OT) was carried out as pre- maximal relaxation and contraction respectively viously described (Engstrøm et al. 1988a). Plasma (Emax) and the agonist concentration giving half this membranes were incubated for 60 min with concen- effect (EC50) were calculated. Kruskal–Wallis one trations of [3H]OT varying from 0·42 to 24·20 nM way analysis of variance on ranks was used to
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compare groups. Pair-wise comparisons were done by means of Student’s t-test or the Mann–Whitney test.
RESULTS
RT-PCR RT-PCR with the primers used resulted in highly specific PCR-products. Products of 240 bp repre- sented internal standards for the two primer sets whereas the â2AR and the OTR products consisted of 342 bp and 375 bp respectively. Sequencing of both strands of each receptor product confirmed the expected identities. The sequences conformed to the published sequences, GenBank accession number L39264 for â2AR and V15280 for OTR, with the following exceptions. In L39264 nucle- otides 3588 and 3589 are both As whereas we found only one A in this position. Likewise in V15280 the 1. Standard curves showing the proportion nucleotide 374 is a single C whereas we found a between specific mRNA and internal standards as a sequence of CCC corresponding to this nucleotide function of poly-adenylated mRNA used for RT-PCR.
position. Both of these variants occurred immedi- Oxytocin receptor mRNA ( ) and â2AR mRNA ( ). ately downstream of the coding sequences and Values are means&..., n=3. mRNA amounts for therefore did not influence the structure of the gene RT-PCR were subsequently selected well within the products. linear parts of the curves. The ratio between the receptor products and the internal standards showed sigmoid dependencies of the total poly-adenylated mRNA used for RT-PCR times more than low dose treatment (10 µg/h for (Fig. 1). Subsequently mRNA amounts were 2 days). OT treatment tended to increase mRNA selected to give ratios well within the linear parts of levels when compared with controls. However, this the curves. difference was not statistically significant. RT-PCR amplification of mRNA and internal standard RNA showed log-linear dependencies of â AR mRNA the cycle number in a range from 23 to 28 cycles 2 (Fig. 2). The curves of mRNA and internal Neither isoproterenol nor OT treatment affected standard RNA RT-PCR products were parallel myometrial â2AR mRNA values. Myometrial â2AR over this range. mRNA levels were of similar magnitude as OTR By comparing areas of internal standard DNA mRNA (0·40&0·05 vs 0·40&0·04 amol/mg tissue after PCR and internal standard RNA after when vehicles were compared). RT-PCR and taking into account the different number of chains we were able to calculate the Binding of [3H]OT to isolated myometrial efficiency of the RT. This was approximately 90%. plasma membranes The concentration of specific receptor mRNA was in the range of amoles when expressed per mg Isoproterenol treatment for 2 days (10 µg/h) 3 wet tissue weight. Coefficients of variation for increased maximal binding of [ H]OT to isolated isolation of mRNA and RT-PCR HPLC were 6·7% myometrial plasma membranes by 91% (P=0·041, Table 1). Dissociation constants (K ) were not for â2AR and 14% for OTR. d affected by the treatment. OTR mRNA Contractile activity of isolated myometrial Myometrial OTR mRNA results are shown in strips Fig. 3. Isoproterenol treatment for the periods indicated increased mRNA values (P=0·006). High Figure 4 shows dose–response curves of isoproterenol- dose isoproterenol treatment (10 µg/h for 4 days or induced relaxation of uterine strips from rats treated 20 µg/h for 5 days) enhanced OTR mRNA levels 1·5 with isoproterenol or OT. Maximal relaxation
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2. Amplification of cDNA (open symbols) and internal standard DNA (closed symbols) by PCR as function of cycle number. Values are means of three experiments.
1. Kd and Bmax values of the receptor binding of [3H]oxytocin. Values are means&.., n=6
Bmax Kd (nM) (fmol/ìg protein) Treatment Vehicle 1·25&0·39 0·86&0·10 Isoproterenol (10 ìg/h), 2 days 0·95&0·12 1·64&0·29
Plasma membranes were isolated from rats treated with saline or isoproterenol (10 ìg/h) for 2 days.
except when mini-pumps were removed prior to decapitation. OT treatment did not alter maximal isoproterenol- induced relaxation and EC50 values were left unchanged. Since part of the isoproterenol-induced â2- 3. Myometrial OTR mRNA from non-pregnant desensitization theoretically could be a result of an rats treated with OT (0·5 µg/h, 5 days), isoproterenol augmented contractile tone due to endometrial OT (10 µg/h, 2 days), isoproterenol (10 µg/h, 4 days) or liberation, relaxing dose–response curves were also isoproterenol (20 µg/h, 5 days). Values are obtained with OTR blocked by 100 µM atosiban (a means&..., n=3 or 4. competitive OT antagonist) in the organ chamber. Neither strips from isoproterenol treated nor those from untreated rats exhibited altered dose–response decreased following isoproterenol treatment relations in the presence of atosiban (data not (Emax,iso) for 2, 4 or 5 days (P=0·004, Fig. 5). When shown). the osmotic mini-pumps were removed 12 h prior to In Fig. 6 dose–response curves from OT- decapitation of the animals, Emax,iso returned to stimulated uterine strips are shown. Maximal pre-treated levels. EC50 values increased follow- contractile effect (Emax,OT) of OT increased follow- ing isoproterenol treatment in general (P=0·016), ing isoproterenol treatment (106·3&5·5% vs
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4. Dose–response curves from isolated non-pregnant uterine strips challenged with isoproterenol. Rats were pre-treated with OT (0·5 µg/h, 5 days, ★), isoproterenol for 2 days (10 µg/h, ), 4 days (10 µg/h, ), 5 days (20 µg/h, ) or vehicle (). In one experiment the pumps were removed 12 h prior to decapitation from rats treated with isoproterenol for 4 days (10 µg/h, ). Points are means&..., n=3 or 4.
86·3&5·0%, P=0·036). EC50 values were left et al. 1994). Lohse et al. (1989) reported that unchanged (0·54&0·08 nM vs 0·80&0·15 nM, inhibition of the â2AR kinase prevented homolo- P=0·157). gous desensitization of â2ARs in human epidermoid cells. Using receptor binding analysis, Dayes & Lye (1990) found a reduction in â2AR concentrations DISCUSSION in non-pregnant myometrium within 1 h of iso- proterenol challenge. In hamsters with hereditary Desensitization to â2-adrenergic agonists has been cardiomyopathy, isoproterenol-stimulated adenylate described in many tissues (Lefkowitz 1979, Dayes & cyclase activity was decreased but direct stimulation Lye 1990, Herman Gnjidic et al. 1994, St Onge of the cyclase with forskolin or fluoride was intact,
Journal of Molecular Endocrinology (1998) 20, 261–270
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5. Emax and EC50 values obtained from dose–response curves from isolated non-pregnant uterine strips challenged with isoproterenol. Rats were pre-treated with OT (0·5 µg/h, OT), isoproterenol for 2 days (10 µg/h, iso2), 4 days (10 µg/h, iso4), 5 days (20 µg/h, iso5) or vehicle. In one experiment the pumps were removed 12 h prior to decapitation from rats treated with isoproterenol for 4 days (10µg/h, iso4rem). Values are means&..., n=3 or 4.
of non-pregnant estrogen-primed rat myometrium in terms of Emax,iso approximated 50%. The process was reversed when the agonist stimulation was withdrawn prior to decapitation of the animals. Since Emax,iso and EC50 values of isolated uterine strips challenged with isoproterenol decreased and increased respectively following long-term infusion of isoproterenol, desensitization appears to involve both a reduction in the number of active receptors and impairment of the coupling between receptor occupancy and contractile response. Further our results showed that â2AR mRNA values remained unchanged following long-term isoproterenol treat- ment in vivo. This suggests that myometrial â2AR regulation does not occur at the level of gene expression unless an altered transcription rate following isoproterenol challenge is counter- regulated by changes in the stability of mRNA. Other authors have found both up- and down- 6. Dose–response curves of isolated regulation of â2AR mRNA following isoproterenol non-pregnant uterine strips stimulated with OT. Rats challenge. In DDT1 MF-2 hamster vas deferens were pre-treated with isoproterenol for 2 days (10 µg/h, cells in culture Hadcock & Malbon (1988) and ) or vehicle ( ). Points are means&..., n=4. Hadcock et al. (1989) observed a time- and dose- dependent decrease in â2-adrenergic responsiveness following isoproterenol incubation. This was ac- suggesting that â-desensitization reflects perturba- companied by a reduction in â2AR mRNA levels. tion of the receptor-mediated stimulation (St Onge On the other hand, in similar cell cultures Collins et al. 1994). In the present study â2-desensitization et al. (1989) found that short-term exposure to
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epinephrine stimulated the rate of â2AR gene tachyphylaxis to â2-adrenomimetics following â2AR expression resulting in steady state levels of mRNA stimulation may partly be due to an augmented 3-4 fold higher than in unstimulated cultures. In rat contractile tone imposed by newly synthesized heart cell-line H9c2, isoproterenol exposure led to OTR. a 42% down-regulation of â2AR mRNA (Dangel The mechanism behind the induction of OTR by et al. 1996). It thus appears that desensitization to isoproterenol treatment in estrogen-primed rats â2-stimulants involves gene expression to a varying remains unclear. It has been amply demonstrated extent depending on the tissue and species involved. that estrogen in itself up-regulates OTR mRNA Desensitization of the myometrium to OT is far (Breton et al. 1996, Ostrowski & Lolait 1996, Breton less extensively studied than â2AR desensitization. & Zingg 1997, Quinones Jenab et al. 1997) and the We earlier reported that prolonged exposure to OT effect of isoproterenol in the present study may in vivo resulted in a decreased number of OT therefore appear on the basis of an altered biological binding sites in the estrogen-primed non-pregnant half-life of estrogens. We have, however, treated our rat myometrium and that this effect was associated rats with a quite high pharmacological dose of with a reduced maximal contractile response to OT estradiol benzoate. In this context it seems unlikely stimulation (Engstrøm et al. 1988a). Since admin- that a potential change in estrogen secretion rate or istration of the OTR antagonist atosiban increased turnover, induced by isoproterenol, should affect the number of OTRs (Engstrøm et al. 1988a), it is the influence of administered estrogen on the OTR not likely that the desensitization process is a result gene. Jeng et al. (1995) found that activation of of receptor occupancy per se but requires activation cAMP by forskolin increased the number of OTR of the OTR. Similarly Lutz et al. (1992) found in rabbit amnion cells. Although cortisol did not vasopressin V1 receptor internalization in rat alter the ability of isoproterenol to increase intra- smooth muscle only in response to agonist but not cellular cAMP, and cAMP did not change the antagonist binding. In the present paper treatment number of cortisol receptors, the increase in OTR with OT in vivo did not change OTR mRNA was potentiated by cortisol. The authors suggested indicating that the previously described homologous that cAMP affects steroid receptor–DNA inter- myometrial desensitization to OT does not include action thereby increasing OTR gene expression. A regulation at the level of gene expression. similar mechanism may underlie the effect of iso- Heterologous receptor regulation has been proterenol on OTR in estrogen-primed rat myo- described in different tissues. In rat myometrium metrium. cAMP regulates transcription through the Engstrøm et al. (1988b) found that long-term OT cAMP response element (CRE). CRE interacts with treatment down-regulated bradykinin receptors CRE binding protein (CBP). Smith et al. (1996) when evaluated with radioligand binding studies. reported that CBP enhanced estrogen receptor Likewise the up-regulation of several receptors by transcriptional activity 10-fold. Likewise an in- estrogen is well known (Soloff 1975, Potvin & crease in estrogen receptors may augment OTR Varma 1991, Mayes et al. 1996, Parker et al. 1996). expression via estrogen response elements located in We found that isoproterenol infusion in vivo the OTR gene. induced OTR mRNA formation in the non- In summary we have shown that neither pregnant estrogen-dominated rat myometrium. homologous â2AR nor homologous OTR desensi- This effect was accompanied by a 91% rise in the tization in non-pregnant rat myometrium involves number of OTR binding sites and a 23% increased changes in mRNA levels and therefore they appear maximal response of isolated uterine strips to OT. to be post-transcriptional events. Long-term treat- Since OTR mRNA increased more than 200%, we ment with the â2-agonist isoproterenol induced suggest that only a fraction of available mRNA is formation of OTR mRNA, increased the number of processed further down-stream to form plasma OTR and augmented maximal response of isolated membrane associated receptors. The discrepancy uterine strips challenged with OT. We suggest that between Emax,OT of uterine strips and Bmax of the â2AR desensitization in non-pregnant estrogen- receptor binding indicates that part of the newly primed rat myometrium involves modulation of synthesized OTR are spare receptors. Due to the OTR gene expression. unaltered values of EC50 and Kd neither receptor affinity nor the coupling between OTR occupancy and contractile response appears to be affected by ACKNOWLEDGEMENTS isoproterenol infusion. According to the above results â2AR desensitiz- The present study was supported by The ation in the myometrium is accompanied by an Danish Hospital Foundation for Medical Research up-regulation of OTR. Thus the well-known (Region of Copenhagen, The Faeroe Islands and
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