ABSTRACT
An impaired nitrergic system and altered redox signaling contribute to gastric dysmotility in diabetics. Our earlier studies show that NF-E2-related factor 2 (NRF2) and phase II antioxidant enzymes play a vital role in gastric neuronal nitric oxide synthase (nNOS) function. This study aims to investigate whether supplementation of sepiapterin (SEP), a precursor for tetrahydrobiopterin (BH4) (a cofactor of NOS) via the salvage pathway, restores altered nitrergic systems and redox balance in spontaneous diabetic (DB) female rats. Twelve-week spontaneous DB and age-matched, non-DB rats, with and without dietary SEP (daily 20 mg/kg body wt for 10 days) treatment, were used in this study. Gastric antrum muscular tissues were excised to investigate the effects of SEP in nitrergic relaxation and the nNOS-nitric oxide (NO)-NRF2 pathway(s). Dietary SEP supplementation significantly ( P < 0.05) reverted diabetes-induced changes in nNOS dimerization and function; nitric oxide (NO) downstream signaling molecules; HSP-90, a key regulator of nNOSα activity and dimerization; miRNA-28 that targets NRF2 messenger RNA (mRNA), and levels of microRNA (miRNA) biogenesis pathway components, such as DGCR8 (DiGeorge Syndrome Critical Region Gene 8) and TRBP (HIV1-1 transactivating response RNA-binding protein). These findings emphasize the importance of the BH4 pathway in regulating gastric motility functions in DB animals by modulating nNOSα dimerization in association with changes in enteric NRF2 and NO downstream signaling. Our results also identify a new pathway, wherein SEP regulates NRF2 mRNA turnover by suppressing elevated miRNA-28, which could be related to alterations in miRNA biogenesis pathway components. NEW & NOTEWORTHY This study is the first to show a causal link between NF-E2-related factor 2 (NRF2) and neuronal nitric oxide synthase (nNOS) in gastric motility function. Our data demonstrate that critical regulators of the miRNA biosynthetic pathway are upregulated in the diabetic (DB) setting; these regulators were rescued by sepiapterin (SEP) treatment. Finally, we show that low dihydrofolate reductase expression may lead to impaired nNOS dimerization/function-reduced nitric oxide downstream signaling and elevate oxidative stress by suppressing the NRF2/phase II pathway through miRNA; SEP treatment restored all of the above in DB gastric muscular tissue. We suggest that tetrahydrobiopterin supplementation may be a useful therapy for patients with diabetes, as well as women with idiopathic gastroparesis.
Subject(s)
Diabetes Mellitus/drug therapy , Gastrointestinal Motility , NF-E2-Related Factor 2/metabolism , Nitric Oxide Synthase Type I/metabolism , Pterins/therapeutic use , Pylorus/drug effects , Animals , Diabetes Mellitus/physiopathology , Female , HSP90 Heat-Shock Proteins/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Muscle Relaxation , NF-E2-Related Factor 2/genetics , Pterins/pharmacology , Pylorus/metabolism , Pylorus/physiopathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Signal TransductionABSTRACT
This study examined lung resistance protein (LRP) and multidrug resistance protein (MRP) in lung tumour tissue from 92 patients with non-small cell lung cancer (NSCLC) and normal lung tissue from 20 patients with benign lung tumours. The rates for LRP- and MRP-positive tumours among the NSCLC cases were 54% and 59%, respectively, and their combined positive rate was 45%. These rates were significantly higher than in normal lung tissue. The rates of LRP- and MRP-positive tumours were significantly higher among cases of adenocarcinoma than in cases of squamous cell carcinoma, and in highly differentiated tumours compared with tumours of low or moderate differentiation. There was a significant association between LRP- and MRP-positive tumours and a decrease in overall survival. In conclusion, LRP and MRP play a role in multidrug resistance in NSCLC and are related to prognosis in patients with NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Lung Neoplasms/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Vault Ribonucleoprotein Particles/metabolism , Adult , Aged , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Female , Humans , Kaplan-Meier Estimate , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , PrognosisABSTRACT
Sildenafil, the active ingredient in Viagra, has been purified from commercially available tablets. The purification, using Sephadex G25 chromatography under conditions of low ionic strength, is simple and inexpensive. Sildenafil purified according to this protocol has been characterized with respect to its IC50 for PDE5, its ultraviolet absorption profile, and by collision-induced dissociation fingerprinting, positive ion nanospray, and MALDI mass spectrometry. Tritated sildenafil (6 Ci/mmol) was prepared commercially using the sildenafil purified by this protocol and was verified to retain the potency of unlabeled sildenafil. This protocol and similar procedures will allow investigators to easily isolate sufficient amounts of sildenafil or other PDE5 inhibitors for conducting biochemical and in vitro studies of drug action.
