Tachykinins and tachykinin receptors in human uterus

Abstract

(1) Studies were undertaken to determine the nature of the receptors mediating contractile effects of tachykinins in the uteri of nonpregnant women, and to analyse the expression of preprotachykinins (PPT), tachykinin receptors and the cell-surface peptidase, neprilysin (NEP), in the myometrium from pregnant and nonpregnant women. (2) The neurokinin B (NKB) precursor PPT-B was expressed in higher levels in the myometrium from nonpregnant than from pregnant women. Faint expression of PPT-A mRNA was detectable in the myometrium from nonpregnant but not pregnant women. PPT-C, the gene encoding the novel tachykinin peptide hemokinin-1 (HK-1), was present in trace amounts in the uteri from both pregnant and nonpregnant women. (3) Tachykinin NK(2) receptors were more strongly expressed in tissues from nonpregnant than from pregnant women. NK(1) receptor mRNA was present in low levels in tissues from both pregnant and nonpregnant women. A low abundance transcript corresponding to the NK(3) receptor was present only in tissues from nonpregnant women. (4) The mRNA expression of the tachykinin-degrading enzyme NEP was lower in tissues from nonpregnant than from pregnant women. (5) Substance P (SP), neurokinin A (NKA) and NKB, in the presence of the peptidase inhibitors thiorphan, captopril and bestatin, produced contractions of myometrium from nonpregnant women. The order of potency was NKA>>SP>/=NKB. The potency of NKA was unchanged in the absence of peptidase inhibitors. (6) The tachykinin NK(2) receptor-selective agonist [Lys(5)MeLeu(9)Nle(10)]NKA(4-l0) was approximately equipotent with NKA, but the tachykinin NK(1) and NK(3) receptor-selective agonists [Sar(9)Met(O(2))(11)]SP and [MePhe(7)]NKB were ineffective in the myometrium from nonpregnant women. (7) The uterotonic effects of [Lys(5)MeLeu(9)Nle(10)]NKA(4-10) were antagonized by the tachykinin NK(2) receptor-selective antagonist SR48968. Neither atropine, nor phentolamine nor tetrodotoxin affected responses to [Lys(5)MeLeu(9)Nle(10)]NKA(4-10). (8) These data are consistent with a role of tachykinins in the regulation of human uterine function, and reinforce the importance of NK(2) receptors in the regulation of myometrial contraction.

Figure 1

Agarose gel showing products of reverse-transcriptase–polymerase chain reaction (RT–PCR) assay for cDNA from uterine samples from (a) a nonpregnant premenopausal woman and (b) a pregnant woman. Single transcripts corresponding to the sizes predicted for the NK₁ receptor (NK₁R), the NK₂ receptor (NK₂R), PPT-B and NEP were detected in all the tissues. The specific bands corresponding to the tachykinin NK₃ receptor (NK₃R) and PPT-A were only detectable in nonpregnant human uteri. The mRNA of PPT-C was not detected after amplification of the usual amount of uterine cDNA. β-actin was used as housekeeping gene. M, molecular size standards. Data are typical of results in five pregnant and five nonpregnant women.

Figure 2

Relative gene expression, obtained by real-time quantitative PCR, of (a) NK₁R; (b) NK₂R; (c) NKB and (d) NEP in cDNA from the uteri of pregnant and nonpregnant women. Values for NK₁R, NK₂R, NKB and NEP mRNA are shown in arbitrary units, relative to β-actin mRNA expression. Each bar represents the mean±s.e.m. of at least 18 uterine cDNA samples from each of four to five nonpregnant and four to five pregnant women. ^*P<0.05, significant difference versus mRNA levels in nonpregnant uteri, Student's t-test for unpaired data.

Figure 3

Representative traces showing contractile activity elicited by the tachykinins (a) SP, (b) NKA, (c) NKB, (d) [Sar⁹Met(O₂)¹¹]SP, (e) [Lys⁵MeLeu⁹Nle¹⁰]NKA(4–10) and (f) [MePhe⁷]NKB, together with their corresponding response to KPSS on myometrial preparations obtained from two nonpregnant women. Responses shown in (a)–(c) were on tissue obtained from a 41-year old woman. Responses shown in (d)–(f) were on tissue obtained from a 38-year old woman. Responses to SP, NKA and NKB are in the presence of peptidase inhibitors thiorphan (3 μM), captopril (10 μM) and bestatin (10 μM).

Figure 4

Log concentration–response curves to tachykinin peptides on myometrial preparations from nonpregnant women. Data points are mean responses±s.e.m. and are expressed as a percentage of the response to KPSS. (a) Mean responses to SP, NKA and NKB in the presence of the peptidase inhibitors thiorphan (3 μM), captopril (10 μM) and bestatin (10 μM) (n=6). (b) Mean responses to [Sar⁹Met(O₂)¹¹]SP, [Lys⁵MeLeu⁹Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB (n=5–6).

Figure 5

Log concentration–response curves to [Lys⁵MeLeu⁹Nle¹⁰]NKA(4–10) in the absence and presence of 1, 3 and 10 nM SR48968 on myometrial preparations from nonpregnant women. Data points are mean responses±s.e.m. and are expressed as a percentage of the response to KPSS (n=5–7).

Figure 6

Log concentration–response curves to NKA in the absence and presence of thiorphan (3 μM), bestatin (10 μM) and thiorphan (3 μM) in combination with bestatin (10 μM) on myometrial preparations from nonpregnant women. Data points are mean responses±s.e.m. and are expressed as a percentage of the response to KPSS (n=5).

Figure 7

Log concentration–response curves to [Lys⁵MeLeu⁹Nle¹⁰]NKA(4–10) in the absence and presence of atropine (0.3 μM), phentolamine (1 μM) and tetrodoxin (1 μM) on myometrial preparations from nonpregnant women. Data points are mean responses±s.e.m. and are expressed as a percentage of the response to KPSS (n=5–6).