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Centro de Investigaciones Científicas Isla de La Cartuja, Instituto de Investigaciones Químicas, 41092 Sevilla, Spain; and Departamento de Genética, Facultad de Biología, Universidad de La Laguna, 38206 Tenerife, Spain
Address all correspondence and requests for reprints to: Dr. Francisco M. Pinto, Instituto de Investigaciones Científicas Isla de La Cartuja, Instituto de Investigaciones Químicas, Avenida Americo Vespuccio s/n, 41092 Sevilla, Spain. E-mail: mluz{at}cica.es
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Abstract |
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Introduction |
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SP and NKA have been localized to a population of nerve fibers, mostly of sensory origin, in the female reproductive tract of mammals (13, 14, 15). To our knowledge, there is no evidence supporting the existence of NKB in these sensory nerves. In uteri from estrogen (E2)-treated, virgin rats SP, NKA, and NKB induce contractile responses that seem mainly mediated by activation of NK2R (16, 17). Tachykinin NK1R and NK2R messenger RNAs (mRNAs) are expressed in appreciable amounts in E2-treated uteri, whereas NK3R mRNA is present in trace amounts, only detectable by amplification of large quantities of uterine complementary DNA (cDNA) (17). However, radioligand binding studies demonstrated the presence of NK3R in uteri from rats in the diestrous stage of the estrous cycle (18). Moreover, NK3R mRNA was found in uteri from ovariectomized rats, being present in a higher amount in animals treated with olive oil than in E2-treated animals (19). Little information is available on the mechanisms responsible for regulating tachykinin receptor gene expression. Corticosteroids have been shown to reduce NK1 receptor mRNA in rat pancreatic AR42J cells (20) and human lung (21), suggesting the presence of a negative glucocorticoid-responsive element in this gene. In the present work, we studied the expression of tachykinin NK1R, NK2R, and NK3R in uteri from ovariectomized rats and whether ovarian steroids modulate this expression. We also analyzed the expression and hormonal regulation of the neutral endopeptidase 24.11 (NEP) or enkephalinase, which appears to be the most important enzyme involved in tachykinin degradation in the rat uterus (16, 17). Finally, we studied the mechanical responses evoked by the selective agonists [Sar9Met(O2)11]SP (NK1R selective), [Nle10]NKA-(4–10) (NK2R selective), and [MePhe7]NKB (NK3R selective) in E2- or P4-treated uteri and correlated the changes in tachykinin receptor mRNA expression with the changes in myometrial contractility after selective activation of NK1R, NK2R, or NK3R.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA isolation
Total RNA from approximately 20 mg uterine tissue was
isolated according to the method of Chomczynski and Sacchi (22). The
RNA pellet was resuspended in 50 µl ribonuclease-free water and
quantified by spectrophotometric measurement at 260 nm. The RNA samples
were then denatured and separated on 1% agarose gels containing 0.66
M formaldehyde. Ethidium bromide-stained 28S and 18S
ribosomal RNA bands were visualized under UV illumination to ensure the
integrity of each RNA preparation. To eliminate contaminating genomic
DNA, total RNA samples were treated with fast protein liquid
chromatography pure deoxyribonuclease I (DNase I; Pharmacia Biotech, Uppsala, Sweden). RNA samples (10 µg each) were
incubated at 37 C for 10 min with 10 U DNase I in DNase buffer [40
mM Tris-HCl (pH 7.5), 6 mM MgCl2,
and 10 U ribonuclease inhibitor RNasin; Promega Corp.,
Madison, WI]. The reaction was stopped by extraction with
phenol-chloroform (1:1, vol/vol), and RNA samples were ethanol
precipitated, dried, resuspended in diethylpyrocarbonate-treated water,
and stored at -70 C until use.
RT-PCR studies
First strand cDNA was synthesized using Moloney murine leukemia
virus reverse transcriptase and random hexamers according to
Pharmacia’s instructions (first strand cDNA synthesis kit,
Pharmacia Biotech) in a 15-µl volume reaction containing
5 µg DNase-treated total RNA. PCR was used to detect the mRNA types
for NK1R, NK2R, NK3R, or NEP using
specific oligonucleotide primers for each one. Amplification of the
ß-actin gene transcript was used to control the efficiency of RT-PCR
among the samples. Primers designed for tachykinin receptors and NEP,
their sizes, and appropriate references are shown in Table 1
. Sequences of sense and antisense
primers for ß-actin were 5'-CCTAGCACCATGAAGATCAA-3' and
5'-TTTCTGCGCAAGTTAGGTTTT-3', respectively (23, 24). The expected size
of the PCR product was 227 bp. PCR mixes contained 0.2 µM
primers, 1.5 U Taq polymerase (Pharmacia Biotech), the buffer supplied, 2.5 mM
MgCl2, 200 µM deoxy-NTP's, and cDNA in 25
µL. Each experiment also contained two negative controls, one with
the RT reaction containing no added RNA and the other one containing
RNA that had not been reverse transcribed. PCR reactions were carried
out using a thermocycle programmable heating block (Pharmacia Biotech). After heating at 94 C for 2 min, the parameters used
for PCR amplification were as follows: denaturation, 30 s at 94 C;
annealing, 30 s at 58 C; and extension, 30 s at 72 C. Cycle
numbers were 35 for tachykinin receptors, 36 for NEP, and 24 for
ß-actin. PCR products were separated by gel electrophoresis on 1.7%
agarose, stained with ethidium bromide, and visualized and photographed
under a UV transilluminator (Photodyne, New Berlin, WI).
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In some experiments, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used instead of ß-actin as the internal standard. The sense and antisense primers employed were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. The number of cycles for PCR amplification was 30, and the size of the PCR product was 452 bp. After normalization to GAPDH mRNA levels, semiquantitative RT-PCR studies were carried out as described above.
DNA sequence analysis
Nucleotide sequences were determined using the fmol sequencing
system (Promega Corp.), as previously described (27).
Functional studies
Longitudinal strips of uterine smooth muscle (8–10 mm in length
and 1–2 mm in diameter) were prepared and mounted in siliconized (with
5% dimethyl-dichlorosilane in chloroform; Sigma Chemical Co., St. Louis, MO) tissue baths containing 4 mL physiological
salt solution of the following composition: 154 mM NaCl,
5.6 mM KCl, 1.9 mM CaCl2, 0.95
mM MgCl2, 5.95 mM
NaHCO3, and 2.78 mM glucose, gassed with 95%
O2-5% CO2. The bathing solution was maintained
at 37 C. Mechanical responses were recorded isometrically by means of
force displacement transducers (FT-03, Grass Instruments, Quincey, MA)
connected to an amplifier (LETICA, Barcelona, Spain) and a recorder (SE
130, ABB Goerz, Zurich, Switzerland). The preparations,
stretched to the optimal resting force of 5 millinewtons, were
equilibrated for 45 min, during which time they were washed with fresh
solution every 15 min. After equilibration, the preparation was induced
to contract two or more times by the administration of a maximally
effective concentration of acetylcholine (ACh; 1 mM) at
30-min intervals until constant responses were obtained. Uterine strips
were then allowed to equilibrate for an additional 60-min period before
challenge with the tachykinin receptor agonists
[Sar9Met(O2)11]SP
(NK1R selective), [Nle10]NKA-(4–10)
(NK2R selective), or [MePhe7]NKB
(NK3R selective; Bachem, Bubendorf, Switzerland).
Only one agonist was tested on each strip. In a previous study we
observed that NK1R undergoes rapid desensitization upon
exposure to the agonists (17). For this reason, a single concentration
of [Sar9Met(O2)11]SP (30
nM) was assayed on each preparation. One noncumulative log
concentration-response curve to [Nle10]NKA-(4–10) or
[MePhe7]NKB was constructed on each uterine strip. Each
agonist concentration remained in contact with the tissue for 5 min,
and the tissue was then washed thoroughly and allowed to rest for 20
min before the addition of the next concentration. Responses to all
tachykinins tested were obtained in the presence of the neutral
endopeptidase inhibitor phosphoramidon
(N-(
-L-rhamnopyranosyl-oxyhydroxyphosphinyl)-L-leucyl-L-tryptophan
sodium salt, Sigma Chemical Co.). A maximally effective
concentration of phosphoramidon (1 µM) (17) was
added to the bath 20 min before the tachykinin analog and reapplied
after wash-out of each agonist concentration. At the end of the
experiment, the preparation was washed repeatedly for 60 min before
application of ACh (1 mM) to check the stability
of tissue contractility. This last response served as an internal
standard for all experiments. Contractile responses were measured as
peak increases in force development
([Sar9Met(O2)11]SP) or as
areas under the force-time curve ([Nle10]NKA-(4–10) and
[MePhe7]NKB) during the 5-min period that each
concentration of an agent was in contact with the preparation. The
responses were expressed as a percentage of the peak increase in force
or in the area under the force-time curve measured during a 5-min
period for ACh (1 mM). To measure the areas, polygraph
tracings were scanned and then processed using the Sigma-Scan software
package (Jandel Scientific Corp., Erkrath, Germany).
Statistical analysis
All values are presented as the mean ± SEM; n
represents the number of different animals. Statistical significance of
differences between two means was assessed using Student’s paired or
unpaired t test. Multiple means were compared by one-way
ANOVA using the Bonferroni multiple comparison test (Instat, version
2.02, GraphPad Software, Inc., San Diego, CA). In
functional studies, P < 0.05 was considered
significant. In RT-PCR studies, P < 0.001 was regarded
as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The identity of PCR
products was confirmed by determination of DNA nucleotide sequences.
This analysis demonstrated that the sequence of the amplified PCR
fragments was identical to those previously reported for the rat brain
NK1R (nucleotide positions 804-1035 bp, according to Refs.
11, 12), the rat stomach NK2R (nucleotide positions
513-1003 bp, according to Ref. 7), and the rat brain NK3R
(nucleotide positions 918-1242 bp, according to Ref. 8). Similarly, the
rat ß-actin-amplified sequence was identical to that previously
published (23). In all experiments, the two negative controls yielded
no detectable products, indicating that 1) all reagents were free of
target sequence contamination; and 2) the RT-PCR products do not come
from contaminating genomic DNA.
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).
Additional experiments were performed in uteri from animals treated
with both E2 and P4. Compared with control
(olive oil-treated) rats, E2 treatment caused a 1.9-fold
increase in NK1R mRNA levels. P4 led to a
3.3-fold decrease when administered alone and suppressed the
E2-induced NK1R mRNA rise in rats treated with
both E2 and P4 (Fig. 2A). Neither E2 nor
P4 produced significant alterations in tachykinin
NK2R mRNA expression (P > 0.001; Fig. 2B).
E2 treatment led to a 14.3-fold decrease in uterine
NK3R mRNA levels (Fig. 2C). P4 was without
effect, if administered alone, and did not influence the
E2-induced decrease in NK3R mRNA (Fig. 2C).
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). Compared with
controls, the decrease observed in rats treated with both
E2 and P4 (1.5-fold) did not reach statistical
significance (P > 0.001; Fig. 3). NEP mRNA expression
levels in E2- plus P4-treated rats were not
significantly different from those in E2-treated rats
(P > 0.001; Fig. 3), but were significantly lower than
mRNA levels in P4-treated rats (P < 0.001;
Fig. 3).
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Functional studies
Figure 4 shows the effects of the
selective tachykinin receptor agonists
[Sar9Met(O2)11]SP,
[Nle10]NKA-(4–10), and [MePhe7]NKB on
myometrial contractility in the presence of phosphoramidon (1
µM). The NK1R-selective agonist
[Sar9Met(O2)11]SP (30
nM) caused uterine contraction in ovariectomized rats
treated with either E2 or P4. Compared with
P4-treated animals, the magnitude of the contractile
response was significantly higher in E2-treated animals
(P < 0.01; Fig. 4A). The selective agonist for
NK2R [Nle10]NKA-(4–10) (1 nM-1
µM) induced concentration-dependent contractile responses
that were virtually identical in uteri from E2- or
P4-treated rats (Fig. 4B). Among the tachykinin agonists
tested, [Nle10]NKA-(4–10) showed the higher
Emax; the maximal contraction was similar in
amplitude to that produced by a maximally effective concentration of
ACh (1 mM). The NK3R-selective agonist
[MePhe7]NKB (1 nM-1 µM)
elicited contractions that were less than 10% of the maximum response
to ACh (1 mM) in uteri from E2-treated rats
(Fig. 4C). [MePhe7]NKB induced greater uterine
contraction in P4-treated animals; the maximal effect was
reached at 10 nM (Fig. 4C). Higher concentrations (30
nM to 1 µM) elicited contractile responses of
decreasing amplitude, probably reflecting receptor desensitization, a
phenomenon that has been previously described for NK3R in
other tissues (29).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three types of tachykinin receptors have been characterized by pharmacological, biochemical, and molecular studies (2, 6). NK1R is the receptor with highest affinity for SP; NK2R is the receptor with highest affinity for NKA, and NK3R is the receptor with highest affinity for NKB-(4–6). In the female reproductive tract, SP and NKA have been shown to be present in virtually all mammalian species examined (13, 14, 15). These neuropeptides could influence myometrial activity as well as other reproductive functions in females (15, 30). However, little is known about the tachykinin receptor types expressed in the uterus, their regulation, and their role in uterine function.
Immunocytochemical studies have shown that numerous SP-positive neurons innervate the myometrium and uterine arteries in mammals, suggesting that the female genital tract is an important target organ for SP (13, 14, 15, 31). This neuropeptide increases uterine blood flow, relaxes vascular smooth muscle, and increases the mechanical and myoelectrical activities of uterine smooth muscle (15, 17, 31). In the rat, SP can modulate the IgE-mediated release of histamine and inflammatory cytokines from uterine mast cells in different ways, depending on the phase of the reproductive cycle (30). SP is the preferred endogenous ligand for tachykinin NK1R, but it is not selective enough and can also activate tachykinin NK2R and NK3R types (6). To our knowledge, the physiological relevance of NK1R in the female reproductive tract has not yet been established. The observation that mRNA encoding tachykinin NK1R is expressed in the rat uterus suggests that NK1R could play a role in mediating uterine responses to tachykinins. Our data indicate that NK1R gene expression was decreased after treatment of ovariectomized rats with P4. These results are consistent with those previously reported by Gorbulev et al. (32), who found that NK1R mRNA levels are reduced during pregnancy in the guinea pig uterus. It has also been reported that P4 selectively and negatively regulates the expression of the NK1R gene in rat pancreatic acinar cells (20). The effect of E2 appears less clear. In rat pancreatic acinar AR42J cells, E2 increased NK1R gene expression (33) or had no effect (20). In our study, E2 raised NK1R mRNA levels by 1.9-fold when administered alone. NK1R-encoding mRNA was decreased by 3.3-fold in rats treated with P4 and by 1.8-fold in rats treated with both E2 and P4. The contractile response evoked by the selective NK1R agonist [Sar9Met(O2)11]SP was significantly higher in E2-treated than in P4-treated animals. Taken together, these results suggest that E2 up-regulates NK1R expression in the rat uterus.
Contractile responses to SP, NKA, and NKB in uteri from noncastrated, virgin rats treated with E2 are mainly mediated by activation of NK2R (16, 17). Radioligand binding studies have also demonstrated the presence of tachykinin NK2R in uteri from either E2-treated or untreated rats (16, 18). Our data extend these observations and show that tachykinin NK2R mRNA is expressed in uteri from ovariectomized rats. The present results also demonstrate that treatment of rats with E2 or P4 had no effect on the uterine level of NK2R mRNA. Adcock et al. (21) showed that NK2R mRNA expression in human lung was unaffected by dexamethasone. These data suggest that the expression of this tachykinin receptor type is not subject to modulation by steroid hormones.
Virtually nothing is known about the role that NKB and NK3R may play in the female reproductive system. To our knowledge, the presence of NKB in the genital tract has not been demonstrated. The NK3R is widely distributed in the central nervous system and is present in smaller amounts in peripheral tissues (1, 8). NK3R mRNA is undetectable or present in trace amounts in E2-treated uteri from noncastrated, virgin rats (17). Barr et al. (18) carried out radioligand binding studies with [125I]Bolton-Hunter eledoisin and found NK3R in uteri from rats in the diestrous stage of the estrous cycle. The present data demonstrate that the NK3R gene is expressed in uteri from olive oil-treated ovariectomized rats. A novel human NK3R homolog has recently been characterized (34, 35). This novel NK3R exhibits a great protein sequence similarity to that of the rat NK3R, with an overall identity of approximately 80%. The NK3R fragment amplified in this study encoded the third extracellular loop, the transmembrane segment VII, and a portion of the cytoplasmic carboxyl-terminal. Within this region, there are 57 different nucleotides between the rat NK3R and the recently described human NK3R homolog. The observation that the deduced nucleotide sequence was identical to that previously reported for the rat brain NK3 receptor confirms that the fragment corresponds to the classical NK3 receptor cloned by Shigemoto et al. (8). Our study shows that the NK3R mRNA level was not affected by P4, but was strongly down-regulated in response to E2 administration. This suggests that E2 has a marked negative regulatory effect on the expression of NK3R in the rat uterus.
Enkephalinase or NEP 24.11 appears to be the most important enzyme involved in tachykinin metabolism in the rat uterus (16, 17). In addition to changes in tachykinin receptor mRNA levels, changes in NEP gene expression may be of physiological importance in modulating tachykinin-induced uterine responses. In our study, NEP mRNA levels were 4-fold higher in P4-treated than in E2-treated animals, suggesting that NEP gene expression in the rat uterus is under hormonal control. If we accept that changes in gene expression correlate with changes in the amount of protein, the increased expression of NEP in uteri under P4 dominance would lead to a higher degradation of tachykinins, thus reducing uterine responsiveness to these neuropeptides. A slower degradation of tachykinins must be expected in E2-dominated uteri.
Steady-state mRNA levels may not correlate with the amount of functional protein produced. The translation efficiency, its processing and turnover, and its transport and translocation to the membrane have to be considered. Therefore, it remains to be determined whether alterations in mRNA expression levels seen in this study result in an altered number of functional tachykinin receptors expressed on the plasma membrane. As mentioned previously, autoradiography and radioligand binding studies have demonstrated the presence of tachykinin NK1R, NK1R, and NK3R in the rat uterus (16, 18). It has also been reported that functional responses evoked by SP or the NK1R agonist SP methyl ester in uterine tissues varied markedly depending on the stage of the estrous cycle (18, 30). We studied the contractions evoked by selective NK1, NK2, and NK3 receptor agonists in uterine strips from the same ovariectomized animals in which mRNA expression levels were determined and observed a marked correlation between variations in myometrial contractility and mRNA abundance for each tachykinin receptor. These data support the hypothesis that changes in steady state receptor mRNA levels are probably accompanied by alterations in the sensitivity of uterine cells to tachykinins. Further studies with the recently characterized antisera specific to NK1R, NK2R, and NK3R (36) would help to clarify this issue.
In the uterus, E2 stimulates the growth of the uterus, causing hyperemia, increased vascular permeability, and edema (37). E2 and P4 regulate the influx of eosinophils and macrophages into the uterus (37). Tachykinins, particularly SP, are thought to act as neurogenic inflammatory factors in local inflammatory responses (38, 39). They produce activation of inflammatory cells, vasodilatation, and increased vascular permeability leading to plasma extravasation and edema formation (38, 39, 40). The present data show that tachykinin NK1R, NK2R, and NK3R are expressed in the rat uterus. This and the observation that tachykinin receptor and neutral endopeptidase gene expression are selectively and differentially regulated by ovarian steroids suggest that tachykinins may participate in some way in the cascade of events occurring in the female genital tract during the estrous cycle and/or pregnancy.
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Footnotes |
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2 Recipient of a fellowship from Consellería de Cultura,
Educació i Ciencia, Generalitat Valenciana, Spain.
3 Recipient of a fellowship from Fundación Ramón Areces,
Spain.
Received June 23, 1998.
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, P < 0.001 (significant difference
from mRNA levels in control rats); 
, P <
0.001 (significant difference from mRNA levels in
E2-treated rats); §, P < 0.001
(significant difference from mRNA levels in P4-treated
rats; all by one-way ANOVA).
