Hachiro Usui

New antihypertensive peptides isolated from rapeseed

Four potent angiotensin converting enzyme (ACE) inhibitory peptides, IY, RIY, VW and VWIS, were isolated from subtilisin digest of rapeseed protein. Among them RIY and VWIS are new peptides with IC(50) 28 and 30 microM, respectively. All isolated peptides lowered blood pressure of spontaneously hypertensive rats (SHR) following oral administration. The maximum effect in the case of RIY was observed 4h after administration, while maximum effect of other peptides on blood pressure occurred 2h after administration. Furthermore, the antihypertensive effect of RIY was observed even in old rats, in which ACE inhibitors become less effective, suggesting that a different mechanism other than ACE inhibition is also involved in lowering blood pressure by this peptide. Subtilisin digest of rapeseed protein also significantly lowered blood pressure of SHR after oral administration of a single dosage 0.15 g/kg, exerting maximum antihypertensive effect 4h after administration. This digest appears promising as a functional food, which may be useful in the prevention and treatment of hypertension.

A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery.

In this report, we deal with the isolation of a novel vasorelaxing peptide from a chymotryptic digest of ovalbumin and its vasorelaxing activities. This peptide is composed of Arg‐Ala‐Asp‐His‐Pro‐Phe (RADHPF) in its sequence, corresponding to residues 359–364 of ovalbumin. This peptide (30–300 μM) exerted a dose‐dependent vasodilation in an isolated mesenteric artery from a spontaneously hypertensive rat which was pre‐constricted by phenylephrine, besides the relaxation being endothelium‐dependent. It is noteworthy that the nitric oxide synthase inhibitor N G‐nitro‐L‐arginine methyl ester inhibited this relaxation, implying involvement of nitric oxide in its mechanism of action. Following oral administration of RADHPF at a dose of 10 mg/kg, the systolic blood pressure in a spontaneously hypertensive rat was significantly lowered.

Isolation and characterization of ovokinin, a bradykinin B1 agonist peptide derived from ovalbumin

A vasorelaxing peptide was purified from a peptic digest of ovalbumin, after three steps of reverse-phase HPLC. The structure of the peptide was Phe-Arg-Ala-Asp-His-Pro-Phe-Leu, which corresponded to residues 358-365 of ovalbumin. The peptide was named ovokinin. Ovokinin showed relaxing activity for a canine mesenteric artery (EC50 = 6.3 microM). The relaxing activity was blocked by the bradykinin B1 antagonist [des-Arg9] [Leu8]bradykinin, but not by the B2 antagonist Hoe 140. Ovokinin binds to B1 receptors (IC50 = 64 microM). Prostaglandin I2 was released from the artery after ovokinin stimulation as a relaxing factor. Thus, ovokinin is a weak bradykinin B1 agonist peptide derived from food proteins.

Possible role of endothelial thromboxane A2 in the resting tone and contractile responses to acetylcholine and arachidonic acid in canine cerebral arteries.

Summary The amount of immunoreactive thromboxane B2 (TXB2) released from isolated canine arteries was determined by radioimmunoassay. The amount of TXB2 released from the cerebral, coronary, mesenteric, and saphenous arteries was 47.0 ± 7.2, 4.0 ± 0.6, 4.9 ± 0.5, and 2.7 ± 0.4 pg/mg wet weight tissue/30 min, respectively. The release of TXB2 from the cerebral artery was decreased to <5% by the administration of indomethacin (10-5 M) or OKY-046 (10-4 M), and by intimal rubbing. The release of TXB2 was enhanced nearly twofold by the addition of arachidonic acid (AA) (10-5 M) to the medium, but not by the addition of acetylcholine (ACh) (10-6 M). The cerebral arterial strips maintained the resting tone, which was reduced maximally by papaverine (10-4 M). The resting tone was also reduced dose dependently by a cyclooxygenase inhibitor (indomethacin), a thromboxane A2 (TXA2) synthetase inhibitor (OKY-046), and a TXA2 antagonist (ONO-3708). The resting tone of rubbed strips was about half that of intact strips. ACh and AA induced similar transient contractions in the cerebral artery. Contractions produced by these agents were attenuated by indomethacin (10-7 M), aspirin (5 × 10-5 M), OKY-046 (10-6 M), and ONO-3708 (10-8 M), and abolished by intimal rubbing. From these results, it is concluded that TXA2 is produced in the endothelial cells and may be involved in maintaining the resting tone and contractile response to AA in the canine cerebral artery.

Design of a Highly Potent Anti-hypertensive Peptide Based on Ovokinin(2-7)

Ovokinin(2-7) (RADHPF), an orally active anti-hypertensive peptide derived from ovalbumin, lowers blood pressure in SHRs at a dose of 10 mg/kg. Attempts were made to potentiate its anti-hypertensive activity by replacing the amino acid residues in [Pro2, Phe3]-ovokinin(2-7), which was previously reported to have 33-fold stronger activity than ovokinin(2-7). The anti-hypertensive activity of [Pro2, Phe3]-ovokinin(2-7) was improved by replacement of the C-terminal Phe residue with Trp. Then, the best amino acid residues at other positions for the anti-hypertensive effect were selected. RPLKPW, the most potent derivative obtained, showed significant anti-hypertensive activities at a dose of 0.1 mg/kg after oral administration in spontaneously hypertensive rats (SHRs). Thus, RPLKPW showed 100-fold more potent anti-hypertensive activity than ovokinin(2-7).

Endothelium-dependent contraction induced by nicotine in isolated canine basilar artery -possible involvement of a thromboxane A2 (TXA2) like substance

The present experiments were undertaken to determine whether the response to nicotine in the isolated canine cerebral artery is endothelium-dependent. Changes in the tension of arterial strips were recorded isometrically. Removal of the endothelium was carried out by gentle rubbing, and confirmed by scanning electron microscopy. Rubbing procedure did not affect the contractile response of the strips to serotonin. Treatment of unrubbed strips with nicotine (10(-4)M) caused a transient contraction. This response was abolished by removal of endothelium and attenuated by hexamethonium (5 x 10(-6)M) and atropine (10(-6)M). The nicotine-induced contraction was attenuated also by aspirin (5 x 10(-5)M), a cyclooxygenase inhibitor, OKY-046 (5 x 10(-5)M), a thromboxane A2 (TXA2) synthetase inhibitor and ONO-3708 (5 x 10(-9)M), a TXA2 antagonist. These results indicate that the nicotine-induced contraction in canine cerebral artery is endothelium-dependent, and suggest that the endothelium-derived contracting factor (EDCF) in the nicotine-induced response is a TXA2-like substance.

Studies on the ileum-contracting mechanisms and identification as a complement C3a receptor agonist of oryzatensin, a bioactive peptide derived from rice albumin

Oryzatensin (Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg) is an ileum-contracting and immunostimulating peptide derived from rice albumin. The mechanisms for the ileal contraction that it induces, consisting of rapid and slow components, were examined. The rapid contraction was mediated by histamine release and the slow contraction by a prostaglandin E2-like substance, judging from the effects of various pharmacological inhibitors and antagonists on ileal contraction and titration of histamine release. The contractile profile was very similar to that of human complement C3a(70-77), which is the COOH-terminal octapeptide of C3a and has, although less potent, qualitatively the same biological activities as C3a. Oryzatensin showed homology with C3a(70-77) and affinity for C3a receptors (IC50 = 44 microM) by radioreceptor assay. This is the first report of a food-derived bioactive peptide acting through complement C3a receptors.

Characterization of β-Lactotensin, a Bioactive Peptide Derived from Bovine β-Lactoglobulin, as a Neurotensin Agonist

β-Lactotensin (β-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine β-lactoglobulin. The ileum-contracting activity of β-LT was blocked by the NT1 antagonist SR48692. β-LT was selective for the neurotensin NT2 receptor while neurotensin was selective for the NT1 receptor. β-LT is the first natural ligand showing selectivity for the NT2 receptor. β-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 β-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 β-LT. These results suggest that the hypertensive activity of β-LT is mediated by the NT2 receptor. It was concluded that the NT1 and NT2 receptors mediate the opposite effect on blood pressure.

Identification of casoxin C, an ileum-contracting peptide derived from bovine κ-casein, as an agonist for C3a receptors

Casoxin C (Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg) is a bioactive peptide that was isolated from a tryptic digest of bovine kappa-casein as an anti-opioid peptide in longitudinal strips of guinea pig ileum. Casoxin C also evokes contraction of the ileal strips, and we found that this process was biphasic with rapid and slow components. The contractile profile was very similar to that of human complement C3a(70-77), which is the COOH-terminal octapeptide of C3a and has, although less potent, qualitatively the same biological activities as C3a. Casoxin C also has homology with C3a(70-77). The rapid contraction was mediated by histamine release and the slow contraction was mediated by a prostaglandin E2-like substance, judging from the effects of various pharmacological inhibitors and antagonists on the ileal contraction. Casoxin C had affinity for C3a receptors (IC50 = 40 microM) in the radioreceptor assay. In addition, casoxin C showed phagocyte-stimulating activities. Casoxin C is therefore the first milk-derived peptide identified, that acts through complement C3a receptors.

Endothelium-dependent contraction in intrapulmonary arteries: mediation by endothelial NK1 receptors and TXA2.

1 We have examined whether three natural tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) induce an endothelium‐dependent contraction (EDC) in the rabbit isolated intrapulmonary artery. 2 Removal of the endothelium almost abolished the contraction induced by SP (10−8 m) while it did not attenuate the contraction induced by SP (10−7 m), NKA (10−9‐10−7 m) or NKB (10−9‐10−7 m). 3 The EDC induced by SP (10−8 m) was abolished by NK1 antagonists (FK‐888, CP‐96345, CP‐99994 and SR‐140333) but not by an NK2 antagonist (SR‐48968). 4 The EDC induced by SP was attenuated by cyclo‐oxygenase inhibitors (aspirin and indomethacin), thromboxane A2 (TXA2) synthetase inhibitors (OKY‐046, KY‐234 and KY‐063) and a TXA2 antagonist (S‐1452). 5 The rank order of potency causing endothelium‐independent contraction (EIC) was NKA>NKB>SP. The EIC induced by SP (10−7 m) was attenuated by an NK2 antagonist but not by NK1 antagonists, cyclo‐oxygenase inhibitors, TXA2 synthetase inhibitors or a TXA2 antagonist. 6 In conclusion, SP at 10−8 m induces EDC via endothelial NK1 receptors and TXA2 production, and SP at 10−7 m induces EIC via NK2 receptors in the rabbit intrapulmonary artery.