Inhibition of nitric oxide synthesis potentiates hypertension during chronic glucose infusion in rats.

Endothelial dysfunction has been proposed to contribute to impaired blood flow control or hypertension in many conditions characterized by hyperinsulinemia or hyperglycemia. However, most studies have focused on whether endothelial dysfunction is present in the established phases of these various hypertensive states, and there is little known concerning the role of the endothelium in the initial stages. This study tested whether nitric oxide production, before endothelial dysfunction develops, plays an important role in counteracting the hypertensive response to chronic glucose infusion. Glucose was infused (18.6 mg/kg per minute IV) for 7 days in 8 normal rats (G) and in 9 rats with a long-term background intravenous infusion of N(G)-nitro-L-arginine methyl ester (L-NAME) at 10 microg/kg per minute (G+L). Mean arterial pressure (MAP), measured 24 hours per day, increased an average of approximately 11 mm Hg in the G rats. L-NAME treatment increased MAP an average of 28+/-2 mm Hg in the G+L rats, and glucose infusion raised MAP >30 mm Hg above that, averaging 155+/-8 mm Hg by day 6. In addition, heart rate increased from an average of 389+/-8 bpm to 441+/-16 bpm by day 6, whereas there was no significant change in the G rats. Glomerular filtration rate decreased significantly with L-NAME treatment and decreased in both groups by day 3 of glucose infusion, reaching lower levels in the G+L rats. These results show that NO is required to minimize the increase in MAP during glucose infusion and suggest that renal and neural mechanisms may be important in mediating that effect.

Expression of EphA4 in developing inner ears of the mouse and guinea pig.

The expression of EphA4, an Eph-class receptor tyrosine kinase, was determined by immunohistochemistry in developing inner ears of the mouse and the guinea pig. In the mouse, EphA4 expression was visible in the fibroblasts of the spiral ligament and in the structures that were to become the osseous spiral lamina. Cochlear nerve ganglion cells expressed ephrin-B2, and the modiolus expressed mRNA coding for ephrin-B3, both transmembrane ligands for EphA4. In contrast, in the guinea pig, cells of the cochlear nerve ganglion expressed EphA4, as did supporting cells of the organ of Corti (Hensen's cells and inner pillar cells). There was also some expression in fibroblasts of the spiral ligament but none in the structures that were to become the osseous spiral lamina. It is suggested that in the mouse, EphA4 may help direct the cochlear innervation towards the organ of Corti by a repulsive interaction, but that this is highly species dependent.

A tyrosine kinase screen of mouse vestibular maculae.

Receptor tyrosine kinases allow extracellular signals to influence intracellular events, while other tyrosine kinases are involved in intracellular signalling. They may therefore be involved in the development, maintenance and repair of the sensory epithelia of the inner ear, since these are believed to be affected by inter- and intracellular signalling. In order to analyse possible tyrosine kinases expressed in sensory areas of the inner ear, a reverse transcription polymerase chain reaction screen of microdissected sensory epithelia was undertaken, using primers targeted at conserved sequences in tyrosine kinase domains. Tissue was taken from the maculae of the mouse vestibular organs, and consisted mainly of hair cells and their supporting cells. Of 80 clones sequenced, 49 coded for tyrosine kinases, and 11 for other known molecules. Further analysis of one of the sequences, for FGF receptor 4, showed a novel variant, expressed in the inner ear and elsewhere, with a variation in the intracellular domain which suggests differential activation of known signalling pathways. Other clones coded for tyrosine kinases expected to be involved in cell surface and intracellular signalling. The technique forms a powerful tool for analysing a range of the tyrosine kinases expressed, and provides a starting point for the analysis of cell-cell signalling in the inner ear.

Fibroblast growth factor receptor-4 splice variants cause deletion of a critical tyrosine.

We have identified two novel isoforms of fibroblast growth factor receptor-4 (FGFR4). They result from alternative splicing of intron 17. Two transcripts, both slightly larger than the one coding for the known mouse FGFR4, are generated. The shortest (FGFR4-17a) includes the 31-most 3'-nucleotides of intron 17; the longest (FGFR4-17b) includes all 114 nucleotides of intron 17. Translation of the FGFR4-17a and FGFR4-17b splice variants predicts that both novel putative FGFR4 isoforms have a truncated C-terminal intracellular tail. The first amino acid residue affected by the insertions in both novel isoforms is Tyr-760, a residue that may play a crucial role in intracellular signaling through stimulation of the phosphatidylinositol-biphosphate pathway.

Sequence and tissue distribution of chicken cellular nucleic acid binding protein cDNA.

We sequenced cDNAs coding for chicken cellular nucleic acid binding protein (CNBP). Two slightly different variations of the open reading frame were found, each of which translates into a protein with seven zinc finger domains. The longest transcript contains an in-frame insert of 3 bp. The sequence conservation between chick CNBP cDNAs with human, rat and mouse CNBP cDNAs is extreme, especially in the coding region, where the deduced amino acid sequence identity with human, rat and mouse CNBP is 99%. CNBP-like transcripts were also found in various tissues from insect, shrimp, fish and lizard. Regions with remarkable nucleotide conservation were also found in the 3' untranslated region, indicating important functions for these regions. Quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated that in the chick, CNBP in present in all tissues examined in approximately equal ratios to total RNA. RT-PCR of total RNA isolated from different phyla indicate CNBP-like proteins are widespread throughout the animal kingdom. The extraordinary level of conservation suggests an important physiological role for CNBP.

Sequence of the 5'-flanking and 5'-UTR regions of the rat endothelin-A receptor gene.

Endothelin receptors bind peptides of the endothelin family, the most potent vasoconstrictors known, and have been implicated in hypertension. To begin to define DNA sequences necessary for the transcriptional regulation of the rat endothelin type A receptor (ETA), we have sequenced the 5'-untranslated region (UTR) and part of the 5'-flanking region, a total of 1153 nucleotides. Comparison of the rat and human sequences revealed a 57% similarity in the 5'-flanking sequences, and a 63% similarity in the 5'-UTRs. Several conserved sequences were identified, including GATA and E-boxes, which may be important for the regulation of ETA gene expression. Primer extension analysis identified two transcription initiation sites within the rat gene.