Characterization of beta-lactotensin,a bioactive peptide derived from bovine beta-lactoglobulin,as a neurotensin agonist

beta-Lactotensin (beta-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine beta-lactoglobulin. The ileum-contracting activity of beta-LT was blocked by the NT1 antagonist SR48692. beta-LT was selective for the neurotensin NT2 receptor while neurotensin was selective for the NT1 receptor. beta-LT is the first natural ligand showing selectivity for the NT2 receptor. beta-LT showed hypertensive activity after intravenous administration at a dose of 30 mg/kg in conscious rats, while neurotensin showed hypotensive activity. The hypertensive activity of beta-LT was blocked by levocabastine (1 mg/kg, i.v.), an NT2 antagonist. SR48692, which blocked the hypotensive activity of neurotensin, had no effect on the hypertensive activity of beta-LT. These results suggest that the hypertensive activity of beta-LT is mediated by the NT2 receptor. It was concluded that the NT1 and NT2 receptors mediate the opposite effect on blood pressure.     

Designing of an orally active complement C3a agonist peptide with anti-analgesic and anti-amnesic activity

Complement C3a is an anti-opioid peptide, having anti-analgesic and anti-amnesic effects after intracerebroventricular administration. However, the peptide is inactive after oral administration. Orally active C3a agonist peptide was designed based on the structure of oryzatensin, a C3a agonist peptide derived from rice albumin. Tyr-Pro-Leu-Pro-Arg, a pentapeptide at the carboxyl terminus of oryzatensin is the minimally essential structure for exerting C3a activity. Due to the affinity for mu-opioid receptor, both oryzatensin and Tyr-Pro-Leu-Pro-Arg showed analgesia after intracerebroventricular administration in mice which was blocked by the opioid antagonist naloxone. Tyr-Pro-Leu-Pro-Arg lost opioid activity by substitution the amino terminus tyrosine with other hydrophobic residues. Among the newly designed peptides, Trp-Pro-Leu-Pro-Arg was found to possess the strongest C3a activity. The peptide antagonized morphine-induced analgesia at 300 mg/kg after oral administration and also improved scopolamine- and ischemia-induced amnesia in a step-through passive avoidance test.     

3,4-Dihydroxyphenylethanol (Hydroxytyrosol) Mitigates the Increase in Spontaneous Oxidation of Dopamine During Monoamine Oxidase Inhibition in PC12 Cells

The catecholaldehyde hypothesis predicts that monoamine oxidase (MAO) inhibition should slow the progression of Parkinson’s disease, by decreasing production of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL). Inhibiting MAO, however, diverts the fate of cytoplasmic dopamine toward potentially harmful spontaneous oxidation products, indicated by increased 5-S-cysteinyl-dopamine (Cys-DA) levels. 3,4-Dihydroxyphenylethanol (hydroxytyrosol) is an abundant anti-oxidant phenol in constituents of the Mediterranean diet. Whether hydroxytyrosol alters enzymatic or spontaneous oxidation of dopamine has been unknown. Rat pheochromocytoma PC12 cells were incubated with hydroxytyrosol (10 µM, 180 min) alone or with the MAO-A inhibitor clorgyline (1 nM) or the MAO-B inhibitors rasagiline or selegiline (0.5 µM). Hydroxytyrosol decreased levels of DOPAL by 30 % and Cys-DA by 49 % (p < 0.0001 each). Co-incubation with hydroxytyrosol prevented the increases in Cys-DA seen with all 3 MAO inhibitors. Hydroxytyrosol therefore inhibits both enzymatic and spontaneous oxidation of endogenous dopamine and mitigates the increase in spontaneous oxidation during MAO inhibition.     

Dopamine-derived biological reactive intermediates and protein modifications: Implications for Parkinson's disease

Dopamine (DA) undergoes monoamine oxidase catalyzed oxidative deamination to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is metabolized primarily to 3,4-dihydroxyphenylacetic acid (DOPAC) via aldehyde dehydrogenase (ALDH). Previous studies demonstrated DOPAL to be neurotoxic, more so than DA and other metabolites, and implicated the aldehyde intermediate as a factor in the pathogenesis of Parkinson's disease (PD). However, the mechanism for generation of DOPAL at aberrant levels and the pathways for toxicity are not conclusively known. Various models for DA catabolism revealed the susceptibility of DOPAL biotransformation (e.g., ALDH) to products of oxidative stress, e.g., 4-hydroxy-2-nonenal, at physiologic/pathologic levels and agents that induce oxidative stress. An elevated concentration of DOPAL correlated with increased protein modification with subsequent work demonstrating significant reactivity of the DA-derived electrophile toward protein nucleophiles compared to DA and other metabolites, e.g., DOPAC. The addition of DOPAL to proteins proceeds via reaction of the aldehyde with Lys residues, yielding a Schiff base; however, post-adduction chemistry occurs for the DOPAL-modification resulting in protein cross-linking. Preliminary work indicates enzymes in DA synthesis and catabolism to be cellular targets for DOPAL. Functional consequences for elevated levels of the DA-derived aldehyde and protein modification may include adverse cellular effects. These data implicate DOPAL as a toxic and reactive intermediate potentially serving as a "chemical trigger" for some stage of PD pathogenesis.     

Rubiscolin, a delta selective opioid peptide derived from plant Rubisco.

We found that the sequences YPLDL and YPLDLF in the large subunit of spinach D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) met the structure YP-aliphatic amino acid which might have opioid activity. We then synthesized these peptides to test their opioid activity. The IC(50) of these peptides in mouse vas deferens assay were 51.0 microM and 24.4 microM, respectively, and those in delta receptor binding assay using [(3)H]deltorphin II as radioligand were 2.09 microM and 0.93 microM, respectively. Both peptides were selective for delta receptor. We named them rubiscolin-5 and -6, respectively. Rubiscolin-5 and -6 have antinociceptive activity in mice after i.c.v. or oral administration. The enzymatic conditions to release rubiscolin were investigated using both spinach Rubisco and synthetic fragment peptides. This is the first example of bioactive peptides derived from plant Rubisco.     

Antinociception induced by beta-lactotensin,a neurotensin agonist peptide derived from beta-lactoglobulin,is mediated by NT2 and D1 receptors

In this study, we examined the antinociceptive effect of beta-lactotensin, a neurotensin agonist that has been isolated from the chymotrypsin digest of beta-lactoglobulin as an ileum-contracting peptide. Beta-lactotensin showed naloxone-insensitive antinociceptive activity by the tail-pinch test after i.c.v. (200 nmol/mouse) or s.c. (300 mg/kg) administration in ddY mice. Tolerance was not developed to antinociception induced by beta-lactotensin after repeated s.c. administration for 5 days. The antinociceptive activity of beta-lactotensin was blocked by treatment with the neurotensin NT2 receptor antisense ODN, while treatment with the NT1 receptor antisense ODN had no effect. The antinociceptive activity was also blocked by a dopamine D1 receptor antagonist, SCH23390 (1 microg/mouse, i.c.v.), while a D2 receptor antagonist, raclopride (0.5 microg/mouse, i.c.v.), did not block the activity. These results indicate that the antinociceptive activity of beta-lactotensin is mediated by NT2 and D1 receptors.     

Enzymatic release of neocasomorphin and beta-casomorphin from bovine beta-casein.

Conditions for the release of beta-casomorphin-7 from bovine beta-casein by gastrointestinal proteases in vitro were investigated. beta-Casomorphin-7 was released only from a genetic variant of beta-casein containing a His residue at the 67th position of the peptide chain. Elastase cleaved the peptide bond between Ile66 and His67, releasing the carboxyl terminus of beta-casomorphin-7. Pepsin and leucine aminopeptidase were required to release the amino terminus of this peptide. beta-Casomorphin-9, -13, and -21 also were isolated, and their opioid activities were measured. In this study, we also isolated a novel opioid peptide neocasomorphin-6 (Tyr-Pro-Val-Glu-Pro-Phe), which was released by action of trypsin or pepsin and chymotrypsin.