amastatin and Pain

Endogenous angiotensin (Ang) II and/or an Ang II-derived peptide, acting on Ang type 1 (AT(1)) and Ang type 2 (AT(2)) receptors, can carry out part of the nociceptive control modulated by periaqueductal gray matter (PAG). However, neither the identity of this putative Ang-peptide, nor its relationship to Ang II antinociceptive activity was clarified. Therefore, we have used tail-flick and incision allodynia models combined with an HPLC time course of Ang metabolism, to study the Ang III antinociceptive effect in the rat ventrolateral (vl) PAG using peptidase inhibitors and receptor antagonists. Ang III injection into the vlPAG increased tail-flick latency, which was fully blocked by Losartan and CGP 42,112A, but not by divalinal-Ang IV, indicating that Ang III effect was mediated by AT(1) and AT(2) receptors, but not by the AT(4) receptor. Ang III injected into the vlPAG reduced incision allodynia. Incubation of Ang II with punches of vlPAG homogenate formed Ang III, Ang (1-7) and Ang IV. Amastatin (AM) inhibited the formation of Ang III from Ang II by homogenate, and blocked the antinociceptive activity of Ang II injection into vlPAG, suggesting that aminopeptidase A (APA) formed Ang III from Ang II. Ang III can also be formed from Ang I by a vlPAG alternative pathway. Therefore, the present work shows, for the first time, that: (i) Ang III, acting on AT(1) and AT(2) receptors, can elicit vlPAG-mediated antinociception, (ii) the conversion of Ang II to Ang III in the vlPAG is required to elicit antinociception, and (iii) the antinociceptive activity of endogenous Ang II in vlPAG can be ascribed preponderantly to Ang III.

Topics: Analgesics; Angiotensin II; Angiotensin III; Angiotensin Receptor Antagonists; Animals; Disease Models, Animal; Drug Interactions; Efferent Pathways; Glutamyl Aminopeptidase; Losartan; Male; Microinjections; Neural Inhibition; Nociceptors; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Peptides; Periaqueductal Gray; Rats; Rats, Wistar; Receptors, Angiotensin

Previous in vitro studies have shown that the degradation of [Leu(5)]enkephalin during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Leu(5)]enkephalin administered intra-third-ventricularly on the tail-flick response was increased more than 500-fold by the intra-third-ventricular pretreatment with the three peptidase inhibitors. The antinociceptive effect produced by the [Leu(5)]enkephalin in rats pretreated with any combination of two peptidase inhibitors was significantly smaller than that in rats pretreated with the three peptidase inhibitors, indicating that any residual single peptidase could inactivate significant amounts of the [Leu(5)]enkephalin. The present data, together with those obtained from previous studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play important roles in the inactivation of short endogenous opioid peptides, such as penta-, hepta-, and octa-peptides, administered intra-third-ventricularly to rats.

Topics: Animals; Captopril; Drug Synergism; Enkephalin, Leucine; Glycopeptides; Guinea Pigs; Ileum; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Muscle Contraction; Pain; Peptides; Protease Inhibitors; Rats; Rats, Wistar; Vas Deferens

Previous in vitro studies have shown that the degradation of [Met(5)]enkephalin-Arg(6)-Phe(7) during incubation with cerebral membrane preparations is largely prevented by a mixture of three peptidase inhibitors: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Met(5)]enkephalin-Arg(6)-Phe(7) administered intra-third-ventricularly on the tail-flick response was increased more than 1000-fold by the intra-third-ventricular pretreatment with three peptidase inhibitors. The antinociceptive effect produced by the [Met(5)]enkephalin-Arg(6)-Phe(7) in rats pretreated with any combination of two peptidase inhibitors was significantly smaller than that in rats pretreated with three peptidase inhibitors, indicating that any residual single peptidase could inactivate significant amounts of the [Met(5)]enkephalin-Arg(6)-Phe(7). The present data, together with those obtained from previous studies, clearly show that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play important roles in the inactivation of endogenous opioid peptides, such as [Met(5)]enkephalin, [Met(5)]enkephalin-Arg(6)-Phe(7), [Met(5)]enkephalin-Arg(6)-Gly(7)-Leu(8), and dynorphin A (1-8), administered intra-third-ventricularly to rats.

Topics: Analgesics; Animals; Captopril; Dose-Response Relationship, Drug; Drug Synergism; Enkephalin, Methionine; Glycopeptides; Injections, Intraventricular; Injections, Subcutaneous; Male; Naloxone; Narcotic Antagonists; Pain; Pain Measurement; Peptides; Protease Inhibitors; Rats; Rats, Wistar

Protease-activated receptors (PARs) 1 and 2 are expressed in capsaicin-sensitive sensory neurons, being anti- and pro-nociceptive, respectively. Given the possible cross talk between PAR-2 and capsaicin receptors, we investigated if PAR-2 activation could facilitate capsaicin-evoked visceral pain and referred hyperalgesia in the mouse and also examined the effect of PAR-1 activation in this model. Intracolonic (i.col.) administration of capsaicin triggered visceral pain-related nociceptive behavior, followed by referred hyperalgesia. The capsaicin-evoked visceral nociception was suppressed by intraperitoneal (i.p.) TFLLR-NH2, a PAR-1-activating peptide, but not FTLLR-NH2, a control peptide, and unaffected by i.col. TFLLR-NH2. SLIGRL-NH2, a PAR-2-activating peptide, but not LRGILS-NH2, a control peptide, administered i.col., facilitated the capsaicin-evoked visceral nociception 6-18 h after administration, while i.p. SLIGRL-NH2 had no effect. The capsaicin-evoked referred hyperalgesia was augmented by i.col. SLIGRL-NH2, but not LRGILS-NH2, 6-18 h after administration, and unaffected by i.p. SLIGRL-NH2, and i.p. or i.col. TFLLR-NH2. Our data suggest that PAR-1 is antinociceptive in processing of visceral pain, whereas PAR-2 expressed in the colonic luminal surface, upon activation, produces delayed sensitization of capsaicin receptors, resulting in facilitation of visceral pain and referred hyperalgesia.

Topics: Administration, Rectal; Animals; Behavior, Animal; Capsaicin; Colon; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Hyperalgesia; Injections, Intraperitoneal; Male; Mice; Oligopeptides; Pain; Pain Measurement; Peptides; Receptor, PAR-1; Receptor, PAR-2; Time Factors; Up-Regulation; Viscera

Previous in vitro studies showed that the degradation of dynorphin-(1-8) [dyn-(1-8)] by cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon. In the present investigations, effects of the three PIs on the anti-nociception induced by the intra-third-ventricular (i.t.v.) administration of dyn-(1-8) were examined. The inhibitory effect of dyn-(1-8) on the tail-flick response was increased more than 100-fold by the i.t.v. pretreatment of rats with the three PIs. The inhibition produced by dyn-(1-8) in rats pretreated with any combination of two PIs was significantly smaller than that in rats pretreated with three PIs, indicating that any residual single peptidase could inactivate significant amounts of dyn-(1-8). The antagonistic effectiveness of naloxone, a relatively selective mu-opioid antagonist, indicates that dyn-(1-8)-induced inhibition of tail-flick response in rats pretreated with three PIs is mediated by mu-opioid receptors. Furthermore, mu-receptor-mediated inhibition induced by dyn-(1-8) was significantly greater than that produced by [Met5]-enkephalin in rats pretreated with three PIs. The data obtained in the present investigations together with those obtained in previous studies strongly indicate that dyn-(1-8) not only has well-known kappa-agonist activity but also has high mu-agonist activity.

Topics: Analgesics, Opioid; Analysis of Variance; Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Bacterial Agents; Captopril; Drug Interactions; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glycopeptides; Injections, Intraventricular; Male; Naloxone; Narcotic Antagonists; Pain; Pain Measurement; Peptide Fragments; Peptides; Protease Inhibitors; Rats; Rats, Wistar; Receptors, Opioid, mu

The intra-third-ventricular (i.t.v.) administration of [Met5]-enkephalin (enk) to rats pretreated i.t.v. with three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon, inhibited the tail-flick response. The enk-induced inhibition was augmented by increasing the doses of the three PIs, with the maximum inhibition being attained at the doses of 10 nmol each. The enk-induced inhibition in rats pretreated with any combination of two PIs, however, were markedly smaller than that in rats pretreated with all three PIs, indicating that three kinds of enzymes all played important roles in the inactivation of enk. The inhibitory effect of enk on the tail-flick response in rats pretreated with the three PIs at doses of 10 nmol each was approximately tenfold higher than that of morphine. The relative anti-nociceptive potencies of enk and morphine were similar to the relative inhibitory potencies obtained previously with the isolated guinea pig ileum pretreated with the three PIs, indicating that the hydrolysis of the i.t.v. administered enk was largely prevented by the three PIs. However, the magnitude of the enk-induced inhibition in rats pretreated s.c. with the three PIs indicated that the hydrolysis of enk injected i.t.v. was not largely prevented by the s.c. administration of three PIs at doses up to 10 micromol each/kg.

Topics: Animals; Anti-Bacterial Agents; Area Under Curve; Captopril; Dose-Response Relationship, Drug; Drug Therapy, Combination; Enkephalin, Methionine; Glycopeptides; Injections, Intraventricular; Injections, Subcutaneous; Male; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptors; Pain; Pain Measurement; Peptides; Protease Inhibitors; Rats; Rats, Wistar

The s.c. administration of [Met5]-enkephalin to 10-day-old rats pretreated with the mixture of 3 peptidase inhibitors, amastatin, captopril and phosphoramidon, produced the inhibition of tail-flick response and loss of righting reflex. When infant rats were pretreated with the mixture of any combination of two peptidase inhibitors, however, the change in both the response and the reflex were not produced at all by enkephalin injection, indicating that 3 kinds of enzymes, amastatin-sensitive aminopeptidase(s), captopril-sensitive peptidyl dipeptidase A and phosphoramidon-sensitive endopeptidase 24.11, played an important role in the inactivation of enkephalin after its systemic administration. Additionally, the fact that the two enkephalin-induced effects were more effectively antagonized by naloxone, a relatively selective mu-opioid antagonist, than by naltrindole, a specific delta-antagonist, or by nor-binaltorphimine, a specific kappa-antagonist, showed that these two effects were produced by the interaction of enkephalin with mu receptors. Moreover the involvement of mu receptors in the production of these two effects was shown by the fact that the s.c. administration of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a selective mu agonist, also produced these two effects which were more effectively antagonized by naloxone than by naltrindole or nor-binaltorphimine. Since the magnitude of the two effects induced by enkephalins in 15-day-old rats was significantly lower than that in 10-day-old rats, and the two enkephalin-induced effects were not produced at all in 20-day-old rats, a maturation-induced decrease in the permeability of the blood-brain barrier against opioid peptides was indicated.

Topics: Aging; Animals; Anti-Bacterial Agents; Captopril; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; Female; Glycopeptides; Injections, Subcutaneous; Male; Oligopeptides; Pain; Peptides; Posture; Protease Inhibitors; Rats; Rats, Wistar; Reflex