[des-Arg(1)]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom.

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

Peptidase inhibitor-induced antidiuresis mediated through angiotensin and opioid receptors in the rat hypothalamus.

We examined the effects on urine outflow rate after microinjections of thiorphan (a carboxypeptidase inhibitor) and bestatin (an aminopeptidase inhibitor) into the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei of anesthetized hydrated rats to determine the possible role of neuropeptides in the regulation of urine production. After individual microinjection of the peptidase inhibitors into the nuclei, only thiorphan at 100 nmol administered into the PVN significantly decreased the urine outflow rate. Two consecutive microinjections of the peptidase inhibitors at 100 nmol each into the nuclei induced potent antidiuresis. These effects after microinjections of the peptidase inhibitors into the PVN and SON were diminished by pretreatment with [Sar1,Ile8]angiotensin (ANG) II (an ANG II receptor antagonist) and naloxone (an opioid receptor antagonist) in the PVN and with [Sar1,Ile8]ANG II in the SON, respectively. A vasopressin (AVP) receptor antagonist, d(CH2)5-D-Tyr(Et)VAVP (i.v.), completely blocked the antidiuresis by microinjections of the peptidase inhibitors into both the nuclei. Urinary osmotic pressure was significantly increased by consecutive microinjections of the peptidase inhibitors into the PVN and SON. These results suggest that endogenously-released ANG II and opioid peptides in the PVN and ANG II in the SON regulate urine production mediated through increased AVP release.

Involvement of B2 receptors in the bradykinin-induced relaxation of guinea-pig isolated trachea.

1. The aim of this study was to determine the receptor type and involvement of arachidonic acid metabolites in bradykinin-induced relaxation of the guinea-pig isolated trachea. 2. In the resting tracheal preparation, bradykinin (0.1 nM-30 microM induced a concentration-related contractile response (pD2 = 8.8 +/- 0.3). The maximal tension (1056 +/- 321 mg) was observed at 0.3 microM bradykinin. In contrast, when tracheal preparations were pre-contracted with histamine (30 microM leading to a half-maximum response), a concentration-related relaxation was observed with bradykinin. At the highest concentration of bradykinin used (3 microM), a reversal of 63 +/- 13% of the contractile response to histamine was observed. Both effects of bradykinin were inhibited by the cyclo-oxygenase inhibitor, indomethacin (1 microM). In concentration-response curves, melittin (10 nM-1 microM), a direct activator of phospholipase A2, mimicked both effects of bradykinin. The highest concentration of melittin used (1 microM), induced a tension of 813 +/- 120 mg and led to the reversal of 41 +/- 8% of the contractile response to histamine. The contractile effect of melittin was inhibited in the presence of both indomethacin (1 microM) and AA861 (1 microM), a 5-lipoxygenase inhibitor. 3. [Des Arg9]-bradykinin (1 nM-3 microM), a B1-receptor agonist, was unable to relax precontracted guinea-pig tracheal preparations. The relaxation induced by bradykinin was antagonized by the B2 receptor antagonists, Hoe 140 (D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin) and NPC 17761 (D-Arg0[Hyp3,D-HypE(trans-thiophenyl)7,Oic8]bradykinin ). Hoe 140 (0.1 microM to 0.6 microM) behaved as a non-competitive antagonist with an apparent pA2 = 7.2 +/- 0.4, whereas NPC 17761 (0.3 to 1 microM) competitively antagonized bradykinin-induced relaxation with a pKB = 7.3 +/- 0.2. The Schild regression slope did not differ from unity, 0.96 +/- 0.20, P<0.05.4. These data demonstrate that bradykinin-induced relaxation of guinea-pig trachea occurs via the activation of bradykinin B2-receptors. The stimulation of B2-bradykinin receptors induces the activation of the cyclo-oxygenase pathway, leading either to contraction or relaxation depending on the tone of the trachea.

Effects of thiorphan, bestatin and a novel metallopeptidase inhibitor JMV 390-1 on the recovery of neurotensin and neuromedin N released from mouse hypothalamus.

The effects of the endopeptidase 24.11 ('enkephalinase') inhibitor thiorphan, the aminopeptidase inhibitor bestatin and a novel metallopeptidase inhibitor JMV 390-1 on the K(+)-evoked release of immunoreactive neurotensin and neuromedin N (iNT and iNN) from mouse hypothalamic slices were examined. (JMV 390-1 inhibits several metallopeptidases including endopeptidases 24.11, 24.15 and 24.16, and aminopeptidase N equipotently with Ki values around 50 nM.) Thiorphan increased the recovery of released iNT nearly 2-fold and had no effect on iNN. Bestatin produced a 4-fold increase in iNN recovery and was inactive on iNT. Finally, iNT and iNN recoveries were increased up to 4- and 5-fold, respectively, by JMV 390-1. These results show that in the mouse hypothalamus endopeptidase 24.11 participates with other metalloendopeptidases to the degradation of endogenously released NT while endogenously released NN is principally degraded by aminopeptidase(s).

[Enkephelinase inhibitors acetorphan and SCH34826 potentiate the analgesic effect of low frequency electroacupuncture in rats].

Influence of two novel enkephelinase inhibitors, Acetorphan and SCH34826, on the effect of electroacupuncture analgesia (EAA) has been observed in rats. Administered Acetorphan (2.0-4.0 mg/kg, i.p.) or SCH34826 (30-100 mg/kg, p.o.) potentiated and prolonged the effect of 2 Hz EAA significantly in the nonresponder in which an evident analgesic effect can not be induced by 2 Hz electroacupuncture. This means that che non-responder of 2 Hz EAA can be converted into the responder with these two drugs. The non-responder of 100 Hz EAA, however, can not be changed into the responder with Acetorphan. These results again support our previous reports that enkephelin plays an important role in low frequency EAA. Meanwhile, this suggests us a probable route for potentiating acupuncture analgesia in clinical practice.

Augmented analgesic effects of enkephalinase inhibitors combined with transcranial electrostimulation.

The analgesic effects of very low current transcranial electrostimulation are naloxone-reversible and thus presumably mediated by endogenous opioid activity. The present experiments indicate that blocking enkephalinase activity by i.c.v. thiorphan or i.p. acetorphan results in an increased analgesic effect of electrostimulation as measured by the 50 degrees C wet tail flick test. In the case of each drug, rats receiving both drug and electrostimulation displayed significantly more analgesia than rats receiving electrostimulation and injection vehicle alone, rats receiving drug and sham stimulation or rats receiving vehicle and sham stimulation.