Expression of nitric oxide synthase isoforms in pregnant human myometrium

Abstract

1. Endogenous nitric oxide has been proposed to play a role in the control of myometrial contractility in pregnancy. In this study, the expression, localisation and regulation of nitric oxide synthase (NOS) isoforms have been examined in human pregnant myometrium and cultured human myometrial smooth muscle cells, by immunoblotting, immunohistochemistry and reverse transcription-polymerase chain reaction. 2. Immunoblotting of extracts from freshly isolated myometrial tissue, affinity-enriched for NOS proteins by precipitation with ADP-sepharose, revealed expression of endothelial NOS (eNOS or NOS3) in tissues from preterm, term non-labour and active labour at term. Inducible NOS (iNOS or NOS2) and neuronal NOS (nNOS or NOS1) proteins were not detected at any stage of pregnancy. 3. Immunohistochemical detection showed that expression of eNOS protein was restricted to the endothelium of the myometrial vasculature, with no staining detected in myometrial smooth muscle cells. 4. Messenger RNA for all three NOS isoforms was detected, although iNOS and nNOS mRNAs were detectable only with high cycle number, implying a low copy number. 5. NOS isoforms were not detectable in human myometrial smooth muscle cells cultured from term non-labour pregnancies. Cytokine stimulation of cultured myometrial cells did not induce iNOS expression or nitrite accumulation in the culture medium, although both iNOS protein and nitrite release were detected in the human pulmonary epithelial cell line A549. 6. Levels of eNOS protein and of NOS mRNA expression were not correlated with gestational stage, suggesting that endogenously produced NO is not likely to be a modulator of myometrial tone during human pregnancy.

Figure 1. Immunodetection of NOS isoforms in ADP-sepharose extracts of human pregnant myometrial tissue (MYO) and human myometrial smooth muscle cells (HMSMCs) cultured from term non-labouring myometrium

Representative immunoblots for eNOS (A), iNOS (B) and nNOS (C) with rabbit polyclonal antibodies. D and E are representative blots for eNOS and iNOS obtained with mouse monoclonal antibodies. See Methods for details. Lanes n, i and e: positive controls for nNOS, iNOS and eNOS, respectively. MYO (ADP-sepharose extract of ≈400 mg wet weight per lane): PT, preterm non-labour; T, term non-labour; ALT, active labour at term. HMSMC or, for comparison, A549 epithelial cells (ADP-sepharose extract of ≈3 × 10⁶ cells per lane): C, control unstimulated cells; 24h, cells stimulated for 24 h with 100 μg ml⁻¹ LPS and 10 ng ml⁻¹ each of IL-1β, IFN-γ and TNF-α.

Figure 2. Effect of lipopolysaccharide and cytokines on nitrite accumulation in HMSMCs and A549 epithelial cells

HMSMCs and A549 cells were stimulated with 100 μg ml⁻¹ LPS and 10 ng ml⁻¹ each of IL-1β, IFN-γ and TNF-α. Nitrite accumulation in the culture medium was determined over 24 and 48 h (HMSMCs) and 24 h (A549). +, stimulated cells; -, control unstimulated cells. Determinations on cell cultures from individual uteri were performed in quadruplicate, and values are the means ±s.e.m. of cell cultures from 6 uteri obtained at term in the absence of labour. * P < 0.001 compared with unstimulated HMSMCs and A549 cells.

Figure 3. Immunohistochemical staining of sections of human pregnant myometrial tissue with polyclonal anti-NOS antibodies

Representative sections are shown from preterm and term non-labouring myometrium and from tissue of women in active labour at term. Cryostat sections 5 μm thick were incubated with polyclonal antibodies raised against eNOS, iNOS and nNOS. Negative controls were incubated with non-immune serum. Staining was by avidin-biotin-peroxidase.

Figure 4. Immunohistochemical staining of sections of human pregnant myometrial tissue with monoclonal anti-NOS antibodies

Representative sections are shown of term non-labouring tissue. Cryostat sections 5 μm thick were incubated with monoclonal antibodies raised against eNOS and iNOS. Negative controls were incubated with non-immune mouse IgG. Staining was by avidin-biotin-peroxidase.

Figure 5. RT-PCR for NOS isoforms from human pregnant myometrial tissue (MYO) (A) and HMSMCs (B)

Sequences of eNOS, iNOS and nNOS were amplified from cDNAs derived from preterm non-labour (PT), term non-labour (T) and active labour at term (ALT) myometrial tissue, and from HMSMCs, at the cycle numbers indicated. In B the left-hand lane (MYO) represents pooled cDNAs from myometrial tissue as a positive control. Amplification of a sequence from the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was carried out as a parallel control.