Developmental and regional expression patterns of Type I Nitric Oxide Synthase mRNA and protein in fetal sheep brain during the last third of gestation.

摘要

Type I NOS (nNOS) catalytic activity represents the activity of full-size protein and truncated protein variants originated from many different spliced mRNA variants. Splice mRNA variants are thought to be important in determining the differential organ and subcellular expression of Type I NOS. The present study was directed to increase our understanding of the developmental regulation of Type I NOS in fetal brain. In four discrete areas of the fetal brain, we measured steady-state mRNA levels and catalytic activity and protein mass in the soluble and particulate fractions. Under general anesthesia, we collected sensory-motor cortex, striatum, hippocampus and cerebellum from sheep fetuses at 105, 115, 125 and 135 days gestation (32 fetuses). NOS protein in the soluble and particulate fractions was characterized using Western blot (molecular weight) and arginine to citrulline conversion (enzymatic activity). At the mRNA level, steady state levels were determined using probes directed against exon 2 and exon 21/22 by ribonuclease protection assay (RPA). Our data show that NOS catalytic activity is regulated in a region, subcellular and temporal manner. NOS activity was higher in the soluble fraction in all brain regions and significantly higher levels were found in the soluble fraction of striatum and particulate fraction of hippocampus (P<0.05 by ANOVA). Western blot analysis revealed three distinct molecular weight bands for Type I NOS (155, 144 and 136 kDa). The bands were present in all brain regions and in both cellular compartments with the 155 kDa band being the most abundant molecular form. Truncated protein variants accounted for 25% and 15% of total Type I NOS protein in the soluble fraction and particulate fraction respectively. RPA analysis showed a differential regulation of mRNA variants with exon 2 frame deletions in striatum and hippocampus. A coordinated increase with advancing gestational age of catalytic activity, the full-length protein, the protein variants and steady state mRNA levels was observed in cortex and striatum as demonstrated by higher levels at 125 and 135 days gestation (P<0.05 by ANOVA). NOS enzymatic activity was Ca(2+) and calmodulin dependent. However, in the particulate fraction 20% of the NOS activity was resistant to calmodulin inhibition. In summary, fetal brain Type I NOS mRNA variants are differentially regulated according to brain regions. Our data suggests that exon 2 deleted mRNA variants have low translation efficiency as indicated by the lack of parallel expression of truncated Type I NOS protein variants.