Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33136
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ABSTRACT |
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Calcitonin gene-related peptide (CGRP) inhibits contractions of the myometrium. Isometric force measurements on myometrial strips were carried out to monitor the inhibitory capacity of CGRP in the myometrium during the estrous cycle and in response to estrogen and progesterone in ovariectomized mice. CGRP inhibition of KCl-induced contractions was lowest at estrus and significantly increased during metestrus and diestrus. Progesterone treatment of ovariectomized mice resulted in a significant increase in the responsiveness of the myometrium to CGRP. Expression of CGRP-receptor component protein (CGRP-RCP), a marker of CGRP-receptor expression, was quantitated by Western and Northern blot analyses. The levels of inhibition exerted by CGRP during the various stages of the estrous cycle and in response to steroid hormone treatment correlated with the protein levels of CGRP-RCP. The mRNA levels did not change significantly during the estrous cycle or in response to hormone treatment, indicating that the regulation of CGRP-RCP protein does not occur at the transcriptional level. CGRP had an inhibitory effect both when applied before the stimulus for contraction and when applied during a sustained contracture induced by KCl. This suggests that CGRP-induced generation of second messengers can influence late events in electro-/chemomechanical coupling and/or the contractile machinery directly.
calcitonin gene-related peptide-receptor component protein; receptor activity modifying protein; potassium contracture; smooth muscle
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INTRODUCTION |
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THE SMOOTH MUSCLE LAYER of the uterus, the myometrium, is a marvel of functional adaptation working at extreme opposite ends of the activity spectrum. During pregnancy the myometrium is essentially quiescent, and only weak, asynchronous contractions provide a moderate tonus of the uterine wall. During parturition and postpartum the myometrium generates strong, well-synchronized contractions for expulsion of the fetus and for prevention of postpartum bleeding (42, 51). A similar but less-pronounced change occurs during the estrous and menstrual cycles (26, 38). In the nonpregnant state, the myometrial activity is highest during estrus or the time of ovulation. Activity at this time facilitates sperm entry and migration in the uterus.
The drastic augmentation in myometrial contractility at the time of
parturition is brought about by the upregulation of a series of
stimulatory factors. In most cases the upregulation occurs at the
transcriptional level and is induced by estrogen (6, 27, 36, 55). A
potassium channel presumably involved in pacing of spontaneous
contractions is upregulated at term (6). Both the release of oxytocin
in the uterus (29, 37) as well as the density of myometrial oxytocin
receptors are increased at parturition (16, 24, 41, 50). -Adrenergic
receptors increase in density at term (14, 31, 36). The activity of myometrial cells becomes synchronized at parturition by the acquisition of gap junction channels (12, 13, 17-19, 30, 33, 55). The low
abundance or absence of these factors during pregnancy allows the
uterus to remain quiescent while their appearance at term permits the
uterus to contract vigorously during the birthing process.
Inadvertent activation of any of these factors could result in premature labor. It appears that this is counteracted by a series of inhibitory factors, including calcitonin gene-related peptide (CGRP). Several of the inhibitory factors have also been reported to be regulated in abundance (4, 39, 40, 45, 49, 53). A reciprocal regulation is observed for the stimulatory and inhibitory factors. Thus uterine quiescence during gestation is warranted by the dominance of inhibitory factors. In preparation for birth, the inhibitory factors wane and the stimulatory factors allow the uterus to contract forcefully (4, 6, 13, 18, 24, 29, 36, 39, 40).
Several potential inhibitory factors of myometrial activity have been identified: CGRP (46), relaxin (7), nitric oxide (NO) (8, 11, 40, 49, 53), and carbon monoxide (CO) (1). Some of these inhibitory factors may be part of one signaling pathway. For example, NO has been suggested to mediate CGRP activity (48). Alternatively, they may represent independent pathways. CO generated by heme oxygenase (HO) has only recently been proposed as an inhibitory factor of myometrial activity and is thought to act via cGMP (1). Relaxin has been reported to relax uterine smooth muscle by activation of a calcium-activated potassium channel by a protein kinase A-mediated mechanism (35).
The receptor for CGRP has been elusive. The reason for its resistance to discovery is slowly emerging. It appears that the CGRP receptor belongs to a novel class of receptors in which ligand specificity is not inherent to the standard seven-transmembrane receptor linked to G proteins. Instead, receptor specificity appears to be conveyed onto a standard receptor molecule by a small accessory protein. The first example is CGRP-receptor component protein (CGRP-RCP), a 148-amino acid-containing protein that confers CGRP responsiveness to oocytes expressing this protein exogenously (32). Recently, with the use of the same expression cloning strategy that yielded CGRP-RCP, two more small proteins (RAMP 1 and 2) were identified, which, on interacting with the same seven-transmembrane protein, yield an adrenomedullin receptor or a CGRP receptor (34). During pregnancy the expression levels of CGRP-RCP correlate with the inhibitory activity of CGRP in the myometrium (39), suggesting that CGRP-RCP can be used as a marker for the CGRP receptor.
Most of the changes occurring in the stimulatory factors during gestation and parturition have also been observed at estrus or in response to estrogen (3, 5, 6, 15, 27, 28). Therefore, we investigated in the present study whether the inhibitory effect of CGRP on myometrial contractions also changes during the estrous cycle. In addition, we tested the effect of steroid hormones on CGRP responsiveness of the myometrium. Furthermore, we measured the expression of CGRP-RCP. We also investigated signaling pathways involved in CGRP-mediated relaxation.
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MATERIALS AND METHODS |
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Isometric force measurements of the myometrial strips. Adult female CD1 mice were obtained from Charles River Laboratories (Wilmington, MA). The stage of the estrous cycle was determined by daily monitoring of mice by vaginal smears. Strips of myometrium (0.05-0.07 mm in diameter) were dissected from the longitudinal muscle layer of the uterus of cycling mice at proestrus, estrus, metestrus, and diestrus and were placed in Krebs-bicarbonate solution (in mM: 119 NaCl, 25 NaHCO3, 1.2 MgSO4 3.6 KCl, 1.2 KH2PO4, 2.5 CaCl2, and 11 glucose, pH 7.4). Myometrial strips were mounted between forceps in a 0.5-ml perfusion chamber. Changes in isometric tension were measured by a force-displacement transducer (Grass FT03E) and recorded on a Gould model TA240 recorder. The mounted strips were perfused continuously with gassed (95% O2-5% CO2) Krebs-bicarbonate solution and equilibrated for 30 min before the experiment was started. All measurements were performed at 20°C.
For determination of the inhibitory effect of CGRP, the muscle strips
were first stimulated to contract with a 10-s pulse of high-potassium
Krebs solution (25 mM KCl replacing NaCl). The muscle was then perfused
with 107 M CGRP and subsequently stimulated
repeatedly with 10-s pulses of high-potassium solution.
Hormone administration of ovariectomized animals. Mature female ovariectomized mice (Charles River) were injected intramuscularly with estrogen (20 µg), progesterone (2 mg), or estrogen plus progesterone. Control ovariectomized animals received oil injection. After 24 h, the mice were killed and their uteri excised and used for extracting mRNA and protein as described. Northern and Western blot analyses were carried out to study the effects of hormone treatment on CGRP-RCP mRNA and protein expression. Additionally, isometric force measurements using myometrial strips from hormone-treated ovariectomized animals were carried out to investigate the effects of hormonal treatment on the CGRP responsiveness of the myometrium.
RNA and protein extraction. Uteri from animals at various stages of the estrous cycle were excised and the endometrium removed by scraping. Muscle strips were dissected for force measurements, and the remaining myometrium from individual uteri were placed in Tri Reagent (Molecular Research Center) for both protein and RNA extraction. Control and progesterone-treated ovariectomized mice required pooling of three to four uteri. Tissue samples were homogenized in Tri Reagent (100 mg tissue/1 ml Tri Reagent) using a Polytron homogenizer. Total RNA and protein was isolated according to the manufacturer's instructions (Molecular Research Center).
Poly(A)+ RNA was selected from the total RNA using Poly A Tract mRNA isolation system IV (Promega).
Protein samples were resuspended in 0.1% SDS plus proteinase inhibitors (50 mg/ml lima bean trypsin inhibitor, 2 mg/ml leupeptin, 16 mg/ml benzamidine, 2 mg/ml Pepstatin A), and the protein content was determined using a Pierce Micro-BCA kit (Pierce, Rockford, IL). Both gels and blots were Coomassie stained for visual inspection of protein bands.
Northern blot analysis. Four micrograms of uterine
poly(A)+ RNA extracted from cycling mice (proestrus,
estrus, metestrus, and diestrus) were analyzed by electrophoresis on a
denaturing 1.3% agarose-6% formaldehyde gel, transferred, and
ultraviolet cross-linked onto Nytran nylon membranes (Schleicher & Schuell, Keene, NH). Membranes were hybridized for 16 h with a 148-bp, 32P-labeled mouse CGRP-RCP probe and washed as described
(39). Membranes were exposed to Kodak Biomax film (Eastman Kodak,
Rochester, NY) with an intensifying screen at 80°C.
Membranes were subsequently stripped of probe and reprobed with a
32P-labeled glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probe for normalization purposes. The hybridization signals
were quantified by scanning densitometry, using the Imagequant program
(Molecular Dynamics).
Probes for CGRP-RCP mRNA were synthesized by carrying out PCR on mouse
CGRP-RCP cDNA using primers TCATTGCTGTGAGGAATTCTTGGA and
GAGCAGCGGAAGGAGAGTGGGAAGAAC, in the presence of
[32P]dCTP (3,000 Ci/mmol, New England
Nuclear), with the use of PCR radioactive labeling system (GIBCO BRL).
A cDNA probe for RAMP 1 was prepared similarly with the primers
CTGCCGGGAGCCTGACTATGG and CGATGGTGGACAGCGATGAAG. A PCR-generated
radioactive probe consisting of a 900-bp fragment of GAPDH cDNA was
used for normalization.
Western blot analysis. Protein samples containing 40 µg of protein were resolved by 15% SDS-PAGE at a constant voltage of 140 V and electrotransferred at room temperature at a constant voltage of 15 V overnight to Immobilon-P membranes (Millipore). The transfer buffer contained 25 mM Tris (pH 8.3), 192 mM glycine, and 20% methanol. Following Millipore's rapid immunoblotting protocol, membranes were dried before incubation with primary antibody. Blots were next incubated for 2 h with 1:500 dilution of CGRP-RCP antibody R82, which was raised against the peptide sequence TDLKDQRPRESGKMRHSAG. The blots were washed twice (a minimum of 10 s each) in 0.01 M Tris-buffered saline plus 0.05% Tween (TBS-T) and incubated with a 1:10,000 dilution of anti-rabbit antibody conjugated to horse radish peroxidase (Amersham) for 30 min and washed twice with TBS-T (10 s each). The amount of CGRP-RCP in the membrane preparations was detected by chemiluminescence using SuperSignal Western blotting detection system (Pierce).
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RESULTS |
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Inhibition of myometrial contractions by CGRP. Previous studies of the CGRP effect on myometrial contraction were restricted to observation of inhibition of spontaneous activity or of contractions initiated by stimulatory agonists like ACh. With that approach it is not possible to discriminate between potential targets of CGRP, i.e., whether CGRP inhibits the contractile machinery itself or acts on signaling events. We addressed this issue by using timed applications of a test stimulus in relation to CGRP. As stimulus, we chose elevated potassium concentrations, because exposure of the strips to ACh could not be assumed to result in constant responses, because the ACh receptors may or may not be a variable during the estrous cycle.
Figure 1 shows the effect of
CGRP on potassium-evoked contractions of myometrial muscle strips
isolated from a mouse uterus at metestrus. In Fig. 1A, the
muscle was stimulated with short pulses of potassium every minute
before, during, and after a 12-min lasting exposure to CGRP. The
inhibitory effect of CGRP set in slowly and reached a plateau within
4-5 min. After removal of CGRP it took several minutes for the
contraction amplitudes to recover to the same level as before CGRP
application.
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Contrary to earlier reports (10, 43), potassium-induced contractions of
myometrial muscle strips were inhibited by CGRP even at potassium
concentrations as high as 100 mM (Fig. 1B). Considering that
the inhibitory effect of CGRP requires >1 min to be maximal, it is
possible for the effect to be missed on this basis. Short pulses of
CGRP are as effective in inhibiting myometrial contractions as the
continued presence of the peptide. Figure 2
shows that after a brief pulse of CGRP the amplitudes of
potassium-induced contractions first diminish and then slowly recover
to prepeptide levels with a time course similar to the one shown in
Fig. 1. The observation that the inhibitory effect of CGRP outlasts its presence by many minutes indicates that the signaling pathway initiated
by binding of the peptide to its receptor is slow (>1 min) and long
lasting (>10 min). CGRP not only can prevent generation of full
amplitude contractions but can also relax myometrial muscle that is
already in the contracted state. Figure 3
shows a series of contractures induced by prolonged application of KCl.
CGRP was applied at various time intervals in the continued presence of
KCl. CGRP application relaxed the muscle with a time course similar to
that observed in Figs. 1 and 2.
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Effect of estrous cycle. The response of myometrial muscle
strips isolated from late pregnant mice (before parturition) to CGRP is
fairly constant (39). In contrast, we noticed considerable variability
in myometria of nonpregnant mice. We therefore investigated whether the
CGRP effect is estrous cycle dependent. Uterine muscle strips were
obtained from mice staged by vaginal smears for status in the estrous
cycle. For measuring the inhibitory effect of CGRP on myometrial
contractility, the paradigm shown in Fig. 2 was followed. After a test
contraction induced by 25 mM KCl, 0.1 mM CGRP was applied for 1 min,
followed by a series of 10-s incubations with KCl (Fig.
4). The magnitude of the KCl-induced
contractions did not vary with the estrous cycle.
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The level of inhibition of KCl-induced contractions by CGRP varied
during the estrous cycle. For quantitative analysis, the smallest
contraction in each series (indicated by an asterisk in Fig.
4A) was used to calculate percentage of inhibition
compared with the test contraction (Figs.
4B and 5B). CGRP inhibited
KCl-induced contractions of myometrial strips from mice at metestrus by
>70%. In contrast, during estrus the inhibitory effect of CGRP was
lowest (<30%).
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Dose-response curves of CGRP-mediated inhibition of myometrial
contractile activity at estrus and metestrus have similar
EC50 values but have different maximal levels (Fig.
6).
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CGRP-RCP expression varies during the estrous cycle. We have previously shown that the responsiveness of myometrium to CGRP varies at different gestational stages in correlation with the expression of CGRP-RCP protein levels (39).
The expression of CGRP-RCP protein in the uterus during the estrous cycle was determined by Western blot analysis of uterine protein extracts from myometria of mice at proestrus, estrus, metestrus, and diestrus. Forty micrograms of protein from each stage were subjected to SDS-PAGE and electrotransferred to membranes.
A previously characterized CGRP-RCP antibody, R82, was used for immunodetection of CGRP-RCP protein. CGRP-RCP protein levels were significantly (P < 0.001) lower during estrus compared with metestrus and diestrus (Fig. 5) and correlated with CGRP's capacity to inhibit myometrial contractions.
Northern blot analysis was used to quantify the CGRP-RCP transcript in
the mouse uterus and to monitor any variations in mRNA expression
during the estrus cycle. Poly(A)+ RNA was extracted from
the uteri of animals at proestrus, estrus, metestrus, and diestrus and
hybridized with a 32P-labeled CGRP-RCP-specific probe. A
prominent band of 1.8 kb was detected by hybridization, equally intense
at all stages (Fig. 7). The blot was then
stripped and reprobed for GAPDH expression to determine equal loading
of mRNA. CGRP-RCP mRNA expression did not change significantly
(P > 0.05) during the estrous cycle, as revealed by four
independent Northern blot analyses.
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Effect of progesterone. In rodents, ovaries are the only source
of estrogen and progesterone supply to the uterus. To determine the
effect of hormonal treatment on the CGRP responsiveness of the uterus,
isometric force measurements were carried out. Myometrial strips
obtained from ovariectomized mice that were injected with either
estrogen (20 µg), progesterone (2 mg), or the combination of estrogen
plus progesterone were induced to contract with 25 mM KCl (Fig.
8). The responsiveness of the myometrium of
progesterone-treated animals to CGRP was significantly greater than the
controls (P < 0.001). Preincubation with CGRP inhibited the
KCl-induced contractions by >65% for strips obtained from
progesterone-treated animals. CGRP inhibition of contractions of
myometrial strips obtained from estrogen-treated animals was not
significantly different from the ovariectomized controls. Treatment
with simultaneous combination of estrogen and progesterone also
enhanced the CGRP responsiveness of myometrial strips compared with the
controls (P < 0.05; Fig. 9).
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Forty micrograms of protein isolated from uteri of the ovariectomized animals that were injected with either oil (ovx ctrl), estrogen (ovx E), progesterone (ovx P), or the combination of estrogen and progesterone (ovx E+P) were subjected to SDS-PAGE. Western blot analysis with R82 antibody reveals that the immunoreactive bands of the uterine protein samples from progesterone-treated animals were significantly (P < 0.01) more intense compared with control animals injected with oil (Fig. 9). This indicates that CGRP-RCP immunoreactive protein expression in the uterus is enhanced as a result of progesterone administration. Estrogen treatment did not significantly alter the expression of CGRP-RCP compared with the controls. With hormone treatment, the highest level of CGRP-RCP was correlated with the highest level of inhibition of contraction (Fig. 9).
The change in protein levels does not appear to be a result of transcriptional regulation, because CGRP-RCP mRNA transcription was not altered as a result of hormonal treatment, as revealed by Northern analysis (4 experiments involving pools of 3-4 animals for each experimental condition; data not shown).
Northern blot analysis of myometrial RAMP mRNA. CGRP-RCP is not
the only protein to convey CGRP receptiveness to oocytes. The same
function appears to be exerted by RAMP 1 to form a different CGRP
receptor. We therefore tested whether RAMP 1 expression also correlated
with the inhibitory capacity of CGRP at various stages of the estrus
cycle. Figure 10 shows the hybridization
product of the size reported for RAMP 1 mRNA, but also
shows a larger mRNA species. The intensity of neither
hybridization product was found to correlate with the inhibitory action
of CGRP during the estrus cycle.
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DISCUSSION |
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The present results show that, during the estrus cycle, the sensitivity of the myometrium to CGRP changes. Inhibition of myometrial contractions by the peptide was most pronounced at metestrus and weakest at estrus. Thus, like during gestation and parturition, the regulation of this inhibitory factor of myometrial contraction is reciprocal to that of the stimulatory factors, which include oxytocin receptors, oxytocin release, and gap junctions. This dual regulation of myometrial activity by changing both inhibitory and stimulatory factors warrants quiescence of the pregnant uterus and vigorous contractions for parturition and during estrus. In the case of CGRP, the withdrawal of CGRP-secreting neurons from the uterus at term and during estrus accentuates this effect (2, 20, 56).
The activity of nonpregnant uterus has been used in the past as a reference point for assessment of changes occurring during pregnancy (10). Without staging the estrus cycle, such practice is obsolete, because the changes during the estrus cycle are of the same magnitude as those occurring during pregnancy and parturition.
The mechanism of inhibition of myometrial contractions by CGRP is not clear. Several signaling pathways have been invoked in the literature, including activation of calcium-activated potassium channels, release of nitric oxide, and generation of cAMP.
Activation of adenylate cyclase and generation of cAMP are well-documented consequences of CGRP binding to its receptor (22, 25, 47). Casey and co-workers (9) have shown that CGRP induces a 90-fold increase in cAMP concentration in myometrium. However, no correlated force measurements were performed. Consistent with the cAMP hypothesis is the finding that CGRP can activate the cAMP sensor cystic fibrosis transmembrane conductance regulator in oocytes expressing myometrial mRNA exogenously (39).
Based on patch-clamp data and the observation of attenuation of the inhibitory effect of CGRP on spontaneous contractions by specific channel blockers, Tritthart and co-workers (52) concluded that CGRP inhibits myometrial contractility by opening calcium-activated potassium channels without activation of adenylate cyclase. A similar mechanism but with involvement of adenylate cyclase appears to be established as an operating mechanism in vascular relaxation (44).
Shew et al. (48) reported that NO synthase (NOS) inhibitors can block the relaxing effect of CGRP but not of isoproterenol on the myometrium. Furthermore, they observed that NADPH-diaphorase, thought to be indicative of NOS, is localized in uterine nerve fibers. Based on these observations, the authors concluded that the inhibitory action of CGRP may be dependent on NO formation. NO mediation of CGRP-induced relaxation of vascular smooth muscle has also been reported (2) but appears to be dependent on the presence of the endothelium (54), which probably is the site of NO release. NO, however, may not be involved in potassium channel activation (44).
When assessing the mechanism of action of CGRP in myometrial inhibition, one has to discriminate between inhibition of initiation of contraction (i.e., the electrical signal) vs. inhibition of contraction itself, a distinction often not made and possibly the cause for some confusion in the literature. For example, opening of potassium channels could be very effective in suppressing pacemaker activity and thereby abolishing spontaneous contractions at their origin. Once electromechanical coupling has occurred, opening of potassium channels would become inconsequential. Thus apparently contradictory results may be due to more than one mechanism of action of CGRP in the myometrium. In the present study we found that CGRP attenuates potassium-induced contractions. This observation is inconsistent with an exclusive mechanism involving opening of potassium channels. Because of the change in equilibrium potential associated with increased extracellular potassium ions, opening of more potassium channels would result in a faster depolarization rather than the hyperpolarization that would ensue at physiological potassium concentration (21). Furthermore, CGRP even relaxes contracted muscle, i.e., acts after electromechanical coupling has taken place. Thus during the contraction cycle multiple mechanisms may be activated by CGRP, which could include calcium sequestration and phosphorylation of myosin light chain kinase (23) in addition to potential effects on potassium channels.
The receptor for CGRP at present is not identified with certainty. Several orphan receptors have been proposed to be CGRP receptors with the typical structural feature of seven transmembrane segments. However, functional expression was limited to few cell lines. Xenopus oocytes become responsive to CGRP when they are coerced to exogenously express either one of two small proteins, CGRP-RCP or RAMP 1 (32, 34). These proteins appear to interact with an autochthonous oocyte protein, converting it to an active CGRP receptor. Probably CGRP-RCP and RAMP form different CGRP receptor types. One of the RAMP types (RAMP 2) forms a different receptor altogether, the adrenomedullin receptor. CGRP-RCP appears to mediate the inhibitory effect in the uterus, since this protein but not RAMP 1 shows correlation with inhibitory capacity of CGRP. This correlation was seen both during the estrous cycle (the present study) as well as during pregnancy and parturition (39). CGRP-RCP can, therefore, be used as a marker for CGRP receptor activity.
No changes in mRNA levels corresponding to the variation in protein levels was observed, suggesting that the regulation of CGRP-RCP protein does not occur at the transcriptional level. Yet, progesterone treatment of ovariectomized mice promotes both an increase in CGRP-RCP protein and the inhibitory effect of CGRP on myometrial contractions. It is conceivable that the stability of CGRP-RCP protein is higher when associated with the receptor core protein, which in turn could be regulated at the transcriptional level. Alternatively, progesterone may directly alter translatability or turnover of CGRP-RCP protein.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of General Medical Sciences Grant GM-48610.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Dahl, Dept. of Physiology & Biophysics (R-430), Univ. of Miami School of Medicine, PO Box 016430, Miami, FL 33101 (E-mail: gdahl{at}miami.edu).
Received 4 May 1999; accepted in final form 8 October 1999.
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