Kyotorphin

L-Arginine (L-Arg) is an endogenous substrate for nitric oxide synthase (NOS). In the present study, we examined whether L-tyrosyl-L-Arg (kyotorphin), an endogenous analgesic neuropeptide, might be a substrate for inducible NOS (iNOS) in the brain. Both kyotorphin and L-Arg caused an accumulation of nitrites in lipopolysaccharide (LPS)-treated glial cells cultured from infant rat brains. However, such accumulation of nitrites was not induced by NG-nitro-L-Arg (a NOS inhibitor), L-tyrosyl-D-Arg (D-kyotorphin) or D-Arg. L-Leucyl-L-Arg (an antagonist for kyotorphin receptors) or bestatin (an inhibitor for kyotorphin-hydrolyzing peptidase) did not inhibit the kyotorphin-induced accumulation of nitrites in LPS-treated cells. On the contrary, L-Leucyl-L-Arg caused an accumulation of nitrites in a concentration-dependent manner. The results indicate that nitric oxide (NO) is produced in LPS-treated glial cells directly from kyotorphin through the catalytic action of iNOS.

Kyotorphin (Tyr-Arg) was rapidly degraded in the brain homogenates and purified membrane-bound aminopeptidase from monkey brains. The degradation of kyotorphin by these preparations was effectively inhibited by bestatin. When brain homogenates or slices were incubated with bestatin, kyotorphin was accumulated time-dependently in a rate of 1.0 or 2.1 pmol/mg protein/hr, respectively. The bestatin-induced kyotorphin accumulation was inhibited by leupeptin, p-chloromercuribenzoate, but not phenylmethylsulfonylfluoride or diisopropylphosphate. The kyotorphin accumulation was concentrated in the P2 (crude mitochondrial) fraction, particularly in the particulate or synaptosomal fraction. These findings suggest that kyotorphin may be generated in vitro from precursor proteins by membrane-bound, leupeptin-sensitive "kyotorphin converting enzymes" in close vicinity to membrane-bound aminopeptidase which rapidly degrades kyotorphin generated.

Kyotorphin (KTP; L-tyrosyl-l-arginine), an opioid-like analgesic discovered in the bovine brain, is potentially a neuromodulator because of its localization in synaptosomes, the existence of a specific KTP receptor, and the presence of its biosynthetic enzyme in the brain. KTP is formed in the brain from its constituent amino acids, L-tyrosine and L-arginine, by an enzyme termed KTP synthetase. However, the latter has never been identified. We aimed to test the hypothesis that tyrosyl-tRNA synthetase (TyrRS) is also KTP synthetase. We found that recombinant hTyrRS synthesizes KTP from tyrosine, arginine, and ATP, with Km = 1400 μM and 200 μM for arginine and tyrosine, respectively. TyrRS knockdown of PC12 cells with a small interfering RNA (siRNA) in the presence of 1.6 mM tyrosine, arginine, proline, or tryptophan significantly reduced the level of KTP, but not those of tyrosine-tyrosine, tyrosine-proline, or tyrosine-tryptophan. siRNA treatment did not affect cell survival or proliferation. In mice, TyrRS levels were found to be greater in the midbrain and medulla oblongata than in other brain regions. When arginine was administered 2 h prior to brain dissection, the KTP levels in these regions plus olfactory bulb significantly increased, although basal brain KTP levels remained relatively even. Our conclusion is further supported by a positive correlation across brain regions between TyrRS expression and arginine-accelerated KTP production.

This paper addresses the mechanisms behind selective endothelial permeability and their regulations. The singular properties of each of the seven blood-tissues barriers. Then, it further revisits the physical, quantitative meaning of permeability, and the way it should be measured based on sound physical chemistry reasoning and methodologies. Despite the relevance of permeability studies one often comes across inaccurate determinations, mostly from oversimplified data analyses. To worsen matters, the exact meaning of permeability is being lost along with this loss of accuracy. The importance of proper permeability calculation is illustrated with a family of derivatives of kyotorphin, an analgesic dipeptide.