Characterization of tachykinin receptors in the uterus of the oestrogen-primed rat

Abstract

1. The aim of our study was to characterize the tachykinin receptor population in the oestrogen-primed rat uterus. For this purpose, we investigated the receptor type(s) responsible for tachykinin-induced contraction of longitudinally-arranged smooth muscle layer. The effects of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and several of their analogues with well-defined selectivities for tachykinin NK₁, NK₂ and NK₃ receptors were studied and their inhibition by the selective nonpeptide tachykinin receptor antagonists (S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR 140333, NK₁-selective), (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR 48968, NK₂-selective) and (R)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)- 4-phenylpiperidin- 4-yl)-N- methylacetamide (SR 142801, NK₃-selective) was evaluated. Additionally, expression of tachykinin receptor mRNA was examined by using the reverse transcription-polymerase chain reaction (RT-PCR).

2. SP, NKA, [Nle¹⁰]-NKA(4-10), the analogue with selectivity at the tachykinin NK₂ receptor type, and NKB elicited concentration-dependent contractions of the rat uterus. The pD₂ values were 5.95±0.19; 6.73±0.21; 7.53±0.12 and 5.76±0.21, respectively. The selective agonist for the tachykinin NK₁ receptor [Sar⁹Met(O₂)¹¹]-SP produced a small phasic response in the nanomolar concentration range. The selective tachykinin NK₃ receptor agonist [MePhe⁷]-NKB failed to induce any significant contraction.

3. In the presence of the neutral endopeptidase inhibitor phosphoramidon (1 μM), the log concentration-response curves to exogenous tachykinins and their analogues were shifted significantly leftwards. The pD₂ values were 6.12±0.10, 8.04±0.07, 7.89±0.03 and 6.59±0.07 for SP, NKA, [Nle¹⁰]-NKA(4-10) and NKB, respectively. In the presence of phosphoramidon (1 μM), [Sar⁹Met(O₂)¹¹]-SP (1 nM–0.3 μM) induced concentration-dependent contractions of increasing amplitude when only one concentration of drug was applied to each uterine strip and the pD₂ value was 7.61±0.89. [MePhe⁷]-NKB induced small, inconsistent contractions and, therefore, a pD₂ value could not be calculated.

4. In experiments performed in the presence of phosphoramidon (1 μM), SR 48968 (3 nM–0.1 μM) caused parallel and rightward shifts in the log concentration-response curves of NKA. The calculated pK_B value was 9.16±0.08 and the slope of the Schild regression was 1.28±0.24. SR 48968 (0.1 μM) also antagonized responses to SP with an apparent pK_B value of 7.63±0.13. SR 48968 (0.1 μM) inhibited contractions elicited by NKB (1 nM–3 μM) and [Nle¹⁰]-NKA(4–10) (0.1 nM–3 μM) but had no effect on the response evoked by [Sar⁹Met(O₂)¹¹]-SP (0.1 μM).

5. SR 140333 (0.1 μM) inhibited responses to SP with an apparent pK_B value of 7.19±0.22. This compound did not significantly affect responses to NKA, [Nle¹⁰]-NKA(4-10) and NKB, but suppressed [Sar⁹Met(O₂)¹¹]-SP (0.1 μM)-induced contraction. SR 142801 (0.1 μM) had no effect on responses to natural tachykinins or their analogues.

6. Total RNA was extracted from some of the uteri used in functional studies. RT-PCR assays revealed single bands corresponding to the expected product sizes encoding cDNA for tachykinin NK₁ (587 base pairs) and NK₂ receptors (491 base pairs) (n=6 different animals). A very low abundance transcript corresponding to the 325 base pairs product expected for the tachykinin NK₃ receptor was detected.

7. The present data show that functionally active tachykinin NK₁ and NK₂ receptors are expressed in the oestrogen-primed rat uterus. The NK₂ receptor type seems to be the most important one involved in the contractile responses elicited by tachykinins. NK₃ receptors are present in trace amounts and seem not to be involved in tachykinin-induced contractions.

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