ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species.

Recent studies implicate reactive oxygen species (ROS) such as superoxide anions and H(2)O(2) in the proliferation of systemic vascular smooth muscle cells (SMCs). However, the role of ROS in SMC proliferation within the pulmonary circulation remains unclear. We investigated the effects of endothelin-1 (ET-1), a potential SMC mitogen, on ROS production and proliferation of fetal pulmonary artery SMCs (FPASMCs). Exposure to ET-1 resulted in increases in superoxide production and viable FPASMCs after 72 h. These increases were prevented by pretreatment with PD-156707. Treatment with pertussis toxin blocked the effects of ET-1, whereas cholera toxin stimulated superoxide production and increased viable cell numbers even in the absence of ET-1. Wortmannin, LY-294002, diphenyleneiodonium (DPI), 4-(2-aminoethyl)benzenesulfonyl fluoride, and apocynin also prevented the ET-1-mediated increases in superoxide production and viable cell numbers. Exposure to H(2)O(2) or diethyldithiocarbamate increased viable cell number by 37% and 50%, respectively. Conversely, ascorbic acid and DPI decreased viable cell number, which appeared to be due to an increase in programmed cell death. Our data suggest that ET-1 exerts a mitogenic effect on FPASMCs via an increase in ROS production and that antioxidants can block this effect via induction of apoptosis. Antioxidant treatment may therefore represent a potential therapy for pulmonary vascular diseases.

Role for endothelin-1-induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy.

Our previous studies have demonstrated that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity in vivo and that this inhibition is associated with rebound pulmonary hypertension upon acute withdrawal of inhaled NO. We have also demonstrated that inhaled NO elevates plasma endothelin-1 (ET-1) levels and that pretreatment with PD156707, an ETA receptor antagonist, blocks the rebound hypertension. The objectives of this study were to further elucidate the role of ET-1 in the rebound pulmonary hypertension upon acute withdrawal of inhaled NO. Inhaled NO (40 ppm) delivered to thirteen 4-week-old lambs decreased NOS activity by 36.2% in control lambs (P<0.05), whereas NOS activity was preserved in PD156707-treated lambs. When primary cultures of pulmonary artery smooth muscle cells were exposed to ET-1, superoxide production increased by 33% (P<0.05). This increase was blocked by a preincubation with PD156707. Furthermore, cotreatment of cells with ET-1 and NO increased peroxynitrite levels by 26% (P<0.05), whereas preincubation of purified human endothelial nitric oxide synthase (eNOS) protein with peroxynitrite generated a nitrated enzyme with 50% activity relative to control (P<0.05). Western blot analysis of peripheral lung extracts obtained after 24 hours of inhaled NO revealed a 90% reduction in 3-nitrotyrosine residues (P<0.05) in PD156707-treated lambs. The nitration of eNOS was also reduced by 40% in PD156707-treated lambs (P<0.05). These data suggest that the reduction of NOS activity associated with inhaled NO therapy may involve ETA receptor-mediated superoxide production. ETA receptor antagonists may prevent rebound pulmonary hypertension by protecting endogenous eNOS activity during inhaled NO therapy.

Shear stress regulation of endothelial NOS in fetal pulmonary arterial endothelial cells involves PKC.

We have shown that increased pulmonary blood flow at birth increases the activity and expression of endothelial nitric oxide (NO) synthase (eNOS). However, the signal transduction pathway regulating this process is unclear. Because protein kinase C (PKC) has been shown to be activated in response to shear stress, we undertook a study to examine its role in mediating shear stress effects on eNOS. Initial experiments demonstrated that PKC activity increased in response to shear stress. NO production in response to shear stress was found to be biphasic, with an increase in NO release up to 1 h, a plateau phase until 4 h, and another increase between 4 and 8 h. PKC inhibition reduced the initial rise in NO release by 50% and the second increase by 70%. eNOS mRNA and protein levels were also increased in response to shear stress, whereas PKC inhibition prevented this increase. The stimulation of PKC activity with phorbol ester increased eNOS gene expression without increasing NO release. These results suggest that PKC may play different roles in shear stress-mediated release of NO and increased eNOS gene expression.

A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli.

Most of the sigma (transcriptional initiation specificity) subunits of RNA polymerase, from a wide range of eubacteria, show strong elements of amino acid sequence similarity. There is evidence that two of the "conserved" regions, 2.4 and 4.2, are involved in recognition of the consensus DNA sequences centred near -10 and -35, respectively, which define promoter sites for the initiation of transcription. Since all the alternative sigma subunits of the above type function by binding to a common core polymerase enzyme in a given bacterium, it can be predicted that a hybrid sigma might be functional, and if so should permit RNA polymerase to initiate only at a correspondingly hybrid promoter. To test these predictions, a hybrid gene encoding the amino-proximal 529 amino acids of the major Escherichia coli sigma protein, sigma 70 (including region 2.4) followed by the last 82 amino acids of the heat-shock sigma protein, sigma 32 (including region 4.2) was constructed and fused to Plac on a plasmid. Major-consensus, heat-shock and hybrid promoters were fused to a chloramphenicol acetyl transferase (CAT) reporter gene on a compatible plasmid. CAT assays showed that, as predicted, a promoter with a "heat-shock" -35 consensus and a "major" -10 consensus sequence (PHM) required Plac-dependent production of the hybrid sigma (sigma 70-32) for activity in vivo. PHM then became a strong promoter. The hybrid sigma gene has potential advantages over its parents for structure-function studies.