Suppressive effects by cysteine protease inhibitors on naloxone-precipitated withdrawal jumping in morphine-dependent mice.

The effects of various protease inhibitors on naloxone-precipitated withdrawal jumping were examined in morphine-dependent mice. The doses of morphine were subcutaneously given twice daily for 2 days (day 1, 30 mg/kg; day 2, 60 mg/kg). On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after final injection of morphine (60 mg/kg), and the number of jumping was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by the intracerebroventricular administration of N-ethylmaleimide (0.5 nmol) and Boc-Tyr-Gly-NHO-Bz (0.4 nmol), inhibitors of cysteine proteases involved in dynorphin degradation, 5 min before each morphine treatment during the induction phase, with none given on the test day, as well as by dynorphin A (62.5 pmol) and dynorphin B (250 pmol). However, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, caused no changes. The present results suggest that cysteine protease inhibitors suppress naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation.

Effect of non-selective dopaminergic receptor agonist on disrupted maternal behavior in olfactory bulbectomized mice.

Olfactory bulbectomy (OBX) animals are considered a putative model of depression that produces behavioral, physiological, and neurochemical alterations resembling clinical depression. Depression is a critical cause of child abuse and neglect, and it has been reported that maternal behavior involves dopaminergic neurons of the mesolimbic pathway. In this study, we investigated the effect of apomorphine, a non-selective dopaminergic receptor agonist, on maternal behavior to examine the influence of activated brain dopaminergic function in OBX mice. In addition, we conducted the sucrose preference test to examine the reward system which has a critical relationship to mesolimbic dopaminergic function and maternal behavior. Maternal behavior was observed on postnatal day (PND) 0 and 4. OBX female mice showed a reduction in sucrose preference 2 weeks post surgery. OBX dams showed maternal behavior deficits on PND 0, and these deficits were ameliorated by administration of apomorphine. These results suggest that maternal behavior deficits in OBX dams may involve brain hypodopaminergic function in the central nervous system induced by OBX.

Nociceptive behavior induced by the endogenous opioid peptides dynorphins in uninjured mice: evidence with intrathecal N-ethylmaleimide inhibiting dynorphin degradation.

Dynorphins, the endogenous opioid peptides derived from prodynorphin may participate not only in the inhibition, but also in facilitation of spinal nociceptive transmission. However, the mechanism of pronociceptive dynorphin actions, and the comparative potential of prodynorphin processing products to induce these actions were not fully elucidated. In our studies, we examined pronociceptive effects of prodynorphin fragments dynorphins A and B and big dynorphin consisting of dynorphins A and B, and focused on the mechanisms underlying these effects. Our principal finding was that big dynorphin was the most potent pronociceptive dynorphin; when administered intrathecally into mice at extremely low doses (1-10fmol), big dynorphin produced nociceptive behavior through the activation of the NMDA receptor ion-channel complex by acting on the polyamine recognition site. We next examined whether the endogenous dynorphins participate in the spinal nociceptive transmission using N-ethylmaleimide (NEM) that blocks dynorphin degradation by inhibiting cysteine proteases. Similar to big dynorphin and dynorphin A, NEM produced nociceptive behavior mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site. Our findings support the notion that endogenous dynorphins are critical neurochemical mediators of spinal nociceptive transmission in uninjured animals. This chapter will review above-described phenomena and their mechanism.

Subchronic stress-induced depressive behavior in ovariectomized mice.

AIMS: Mood disorders including depression are more common in women than men, particularly in times of lower estradiol levels. In this study, we investigated the effect of estrogen on emotional behavior in mice in a stress environment. MAIN METHODS: Female mice were divided into four groups: two groups were ovariectomized (OVX) and two were sham-operated. One group each of OVX and sham mice was kept in a normal environment and the other groups were assigned to a daily stress (1 h/day) for 7 days from 5 days after operation. On the 14th day after operation, subjects were measured to assess behavioral specificity, locomotor activity, elevated plus-maze (EPM) behavior, passive avoidance (PA) behavior and forced swimming behavior. KEY FINDINGS: The OVX plus stress (OVX+S) group showed a significant prolongation of immobility compared with the other groups. In all the groups there were no changes in locomotor activity, EPM behavior or PA behavior. We further examined the effect of estrogen against depressive behavior in the OVX+S group. The vehicle or 17beta-estradiol (E2) was administered s.c. to OVX+S mice for 4 days beginning on post-operative day 11. Subchronic E2 treatment decreased the stress response and improved depressive behavior relative to the vehicle group. SIGNIFICANCE: These data have important implications regarding the prevention of depression in postmenopausal women undergoing estrogen therapy.

Involvement of the p53 tumor-suppressor protein in the development of antinociceptive tolerance to morphine.

The present study was designed to determine whether the p53 tumor-suppressor protein is involved in the development of antinociceptive tolerance to morphine. When the doses of morphine (mg/kg per injection) were subcutaneously given into mice as pretreatment twice daily for 2 days (first day (30) and second day (60)), intrathecal (i.t.) administration of morphine (0.1nmol) was inactive due to antinociceptive tolerance in the 0.5% formalin test on the third day. Tolerance to i.t. morphine was significantly suppressed by i.t. injection of pifithrin-alpha (1 and 10nmol), an inhibitor of p53 activation, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-fmk) (1 and 10nmol), a non-selective caspase inhibitor, or N(G)-nitro-l-arginine methyl ester (l-NAME) (2 and 20nmol), a non-selective inhibitor of nitric oxide synthase, 5min before each morphine treatment during the induction, with none given on the test day. Moreover, p53 expression in the spinal cord had increased significantly 14h after the last morphine administration. These results indicate that the increased expression and activation of p53, and the nitric oxide and caspase systems related to p53 may contribute to the development of antinociceptive tolerance to morphine in the mouse spinal cord.

Cysteine protease inhibitors suppress the development of tolerance to morphine antinociception.

The effects of various protease inhibitors on the development of antinociceptive tolerance to morphine were examined in mice. Intrathecal (i.t.) administration of morphine (0.01-1 nmol) produced a dose-dependent and significant antinociceptive effect in the 0.5% formalin test. When the doses of morphine (mg/kg, s.c. per injection) were given as pretreatment twice daily for two days [first day (30) and second day (60)], i.t. administration of morphine (0.1 nmol) was inactive due to antinociceptive tolerance on the third day. Tolerance to i.t. morphine was significantly suppressed by the i.t. injection of N-ethylmaleimide or Boc-Tyr-Gly-NHO-Bz, inhibitors of cysteine proteases involved in dynorphin degradation, as well as by dynorphin A, dynorphin B and (-) U-50,488, a selective kappa-opioid receptor agonist. On the other hand, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, lisinopril, an angiotensin-converting enzyme inhibitor, and phenylmethanesulfonyl fluoride, a serine protease inhibitor, were inactive. These results suggest that cysteine protease inhibitors suppress the development of morphine tolerance presumably through the inhibition of dynorphin degradation.

Differential effects of N-peptidyl-O-acyl hydroxylamines on dynorphin-induced antinociception in the mouse capsaicin test.

In the capsaicin test, intrathecal (i.t.) dynorphins are antinociceptive. Cysteine protease inhibitors such as p-hydroxymercuribenzoate (PHMB) given i.t. augment and prolong their activity. The effect of two novel cysteine protease inhibitors, N-peptidyl-O-acyl hydroxylamines, on the antinociception induced by i.t. administered dynorphin A or dynorphin B has been investigated. When administered i.t. 5 min before the injection of capsaicin (800 ng) into the plantar surface of the hindpaw, dynorphin A (62.5-1000 pmol) or dynorphin B (0.5-4 nmol) produced a dose-dependent and significant antinociceptive effect. The effect of dynorphin A (1 nmol) and dynorphin B (4 nmol) disappeared completely within 180 and 60 min, respectively. PHMB (2 nmol) and Boc-Tyr-Gly-NHO-Bz (BYG-Bz) (2 nmol) co-administered with dynorphin A or dynorphin B significantly prolonged antinociception induced by both. On the other hand, Z-Phe-Phe-NHO-Bz (ZFF-Bz) (1 and 2 nmol) only prolonged antinociception induced by dynorphin A. The results suggest that Z-Phe-Phe-NHO-Bz is an inhibitor of cysteine proteases preferring cleavage of dynorphin A, with less specificity towards dynorphin B in the mouse spinal cord.

Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation.

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-d-aspartate receptor mechanism. Brain Res 2002;952:7-14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

Nociceptive behavior induced by poly-L-lysine and other basic compounds involves the spinal NMDA receptors.

We have previously shown that spermine, a basic polyamine, and big dynorphin, a basic polypeptide, induce nociceptive behavior if injected intrathecally (i.t.) in mice (see [Pain 86 (2000) 55-61] and [Brain Res. 952 (2002) 7-14]). This suggests that other basic molecules might have the same effects. Here, i.t. administration of poly-L-lysine (12 and 36 pg) to mice was found to produce the same characteristic behavioral response, biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 0-10 min after injection. The behavior induced by poly-L-lysine (12 pg) was dose-dependently inhibited by intraperitoneal injection of morphine (0.25-4 mg/kg) and also dose-dependently, by i.t. co-administration of D-(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine hydrogen maleate (MK-801) (0.0156-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 nmol), an antagonist of the polyamine recognition site and the NR2B-containing NMDA receptor subtype. On the other hand, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe7, d-His9]-substance P (6-11), a specific antagonist for substance P (NK1) receptors, or MEN-10,376, a tachykinin NK2 receptor antagonist, had no effect. These results confirm the observations obtained with other basic molecules and suggest that the behavior induced by poly-l-lysine is mediated through the activation of the NMDA receptor ion-channel complex acting either on the polyamine recognition site or on the NR2B subunit.