Leon F. Tseng

Nitric oxide synthase immunoreactivity in the rat, mouse, cat and squirrel monkey spinal cord

The distribution of nitric oxide synthase-immunoreactive neurons was examined in the spinal cord of rats, mice, cats and squirrel monkeys at the light microscopic level. Some sections were processed for choline acetyltransferase immunoreactivity. Double-labeling techniques were used to assess possible co-localization of nitric oxide synthase and choline acetyltransferase immunoreactivity in the same spinal neurons. Nitric oxide synthase-immunoreactive neurons were concentrated in three fairly well-defined regions of the spinal cord of all species studied: (i) the intermediolateral cell column of the thoracic and sacral segments, (ii) lamina X of all segments, and (iii) the superficial layers of the dorsal horn of all segments. A few nitric oxide synthase-immunoreactive neurons were scattered in the deeper laminae and the ventral horn. There were fewer nitric oxide synthase-positive neurons in monkey spinal lamina X and dorsal horn than in similar locations of rodents and felines. Double-staining showed that not all choline acetyltransferase-positive neurons in the intermediate cell column and lamina X were nitric oxide synthase-immunoreactive. In the ventral horn, choline acetyltransferase-positive neurons (presumed motoneurons) were nitric oxide synthase-negative. In addition to cell bodies, nitric oxide synthase-positive fibers were scattered in the dorsal, lateral and ventral horns of all species. Finally, punctate nitric oxide synthase-immunoreactive fibers were seen traversing the dorsal, lateral and ventral white matter, and reaching the respective gray matter. The present study shows that, in spite of quantitative differences, the pattern of distribution of nitric oxide synthase-positive neurons in the spinal cord was similar across the four species. The concentration of nitric oxide synthase-positive neurons in the autonomic nuclei and laminae I, II and X of all four species underscores a prominent role of these neurons in visceral and sensory functions.

Nitric oxide synthase immunoreactivity in rat spinal cord

Immunoreactivity to nitric oxide synthase (NOS-IR) and choline acetyltransferase (ChAT-IR) was detected in the adult rat spinal cord using the avidin-biotin-peroxidase technique. Intensely stained NOS-positive neurons with cell processes were observed in the intermediolateral cell column of the thoracic and sacral segments and around the central canal of all segments. These areas also contained ChAT-IR neurons. A number of small- to medium-sized NOS-IR cells were noted in the superficial and deeper laminae throughout the entire cord. NOS-IR was not detected in the ventral horn motoneurons, which were, however, ChAT-IR. The results indicate that NOS-IR is present in autonomic preganglionic neurons and in selected neurons in the dorsal horn and lamina X, but appears to be absent in motoneurons.

Inhibition of protein kinase C, but not of protein kinase A, blocks the development of acute antinociceptive tolerance to an intrathecally administered μ-opioid receptor agonist in the mouse

A specific protein kinase C inhibitor, calphostin C, which injected alone had no effect on the antinociception induced by intrathecal (i.t.) administration of a selective mu-opioid receptor agonist, [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO), dose-dependently attenuated the development of acute tolerance to the i.t. DAMGO-induced antinociception in male ICR mice. On the other hand, a selective protein kinase A inhibitor, KT5720, did not have any effect on the development of acute tolerance to DAMGO antinociception. These findings suggest that protein kinase C, but not protein kinase A, plays an important role in the development of acute tolerance to the mu-opioid receptor agonist-induced antinociception.

Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

Spinal dynorphin A (1-17) : possible mediator of antianalgesic action

Earlier studies from this laboratory indicated that intracerebroventricular administration of physostigmine and clonidine activated both a spinal descending analgesic and antianalgesic system. It was proposed that the latter was mediated spinally by dynorphin A (1-17), because small intrathecal doses (fmol) of dynorphin A (1-17) antagonized analgesia, while intrathecal administration of naloxone and nor-binaltorphimine (at doses which had no effect on spinal mu and kappa receptors) enhanced analgesia by attenuating the antianalgesic component. In the present studies in mice, using the tail-flick response, intrathecal administration of dynorphin antibody (antiserum to dynorphin) enhanced the analgesic effect of (10 min) physostigmine and clonidine given intraventricularly. Peak effect for the antiserum was at 1 hr. Inhibition of the tail-flick response, induced by DAMGO (Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5, a mu agonist), U50, 488 H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate hydrate, a kappa agonist) and morphine was also enhanced by intrathecal administration of dynorphin antiserum. Thus, a variety of analgesic agonists appear to activate a dynorphin-mediated antianalgesic system. Such a system appears not to be activated by intraventricular administration of beta-endorphin and DPDPE (D-Pen2-D-Pen5-enkephalin, a delta agonist) because neither beta-endorphin- nor DPDPE-induced analgesia was enhanced by intrathecal administration of antiserum. The results of the experiments with the antibody provide further evidence to support the role of dynorphin A (1-17), as a putative endogenous opioid, which mediates an antianalgesic descending system in the spinal cord.

Antinociception Produced by 14,15-Epoxyeicosatrienoic Acid Is Mediated by the Activation of β-Endorphin and Met-Enkephalin in the Rat Ventrolateral Periaqueductal Gray

Cytochrome P450 genes catalyze formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid. The effects of 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET microinjected into the ventrolateral periaqueductal gray (vlPAG) on the thermally produced tail-flick response were studied in male Sprague-Dawley rats. 14,15-EET microinjected into vlPAG (3–156 pmol) dose-dependently inhibited the tail-flick response (ED50 = 32.5 pmol). In contrast, 5,6-EET, 8,9-EET, and 11,12-EET at a dose of 156 pmol were not active when injected into the vlPAG. 14,15-EET failed to displace the radiobinding of [3H][d-Ala2,NHPe4, Gly-ol5]enkephalin (μ-opioid receptor ligand) or [3H]naltrindole (δ-opioid receptor ligand) in crude membrane fractions of rat brain. Tail-flick inhibition produced by 14,15-EET from vlPAG was blocked by intra-vlPAG pretreatment with antiserum against β-endorphin or Met-enkephalin or the μ-opioid receptor antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) or the δ-opioid receptor antagonist naltrindole but not with dynorphin A[1–17] antiserum or the κ-opioid receptor antagonist nor-binaltorphimine. In addition, tail-flick inhibition produced by 14,15-EET treatment was blocked by intrathecal pretreatment with Met-enkephalin antiserum, naltrindole, or CTOP but not with β-endorphin antiserum. It is concluded that 1) 14,15-EET itself does not have any affinity for μ- or δ-opioid receptors and 2) 14,15-EET activates β-endorphin and Met-enkephalin, which subsequently act on μ-and δ-opioid receptors to produce antinociception.

Different types of opioid receptors mediating analgesia induced by morphine, DAMGO, DPDPE, DADLE and β-endorphin in mice

SummaryThe effects of intracerebroventricular (i.c.v.) administration of d-Phe-Cys-Tyr-d-Try-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective mu-opioid receptor antagonist, (Allyl)2-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) and (N,N-Bisallyl-Tyr-Gly-Gly-ψ-(CH2S)-Phe-Leu-OH (ICI 154129), selective delta-opioid receptor antagonists on blocking analgesia induced by β-endorphin, morphine, d-Ala2-NMePhe4-Gly-ol-enkephalin (DAMGO), d-Ala2-d-Leu5-enkephalin (DADLE) and d-Pen2-enkephalin (DPDPE) administered i.c.v. were studied in male ICR mice. The analgesia was assessed by the tail-flick and paw-licking (hot-plate) tests. The potencies of opioid agonists injected i.c.v. for producing analgesia were DAMGO > DADLE > β-endorphin > morphine > DPDPE. Intracerebroventricular administration of CTOP (0.05 μg) selectively antagonized inhibition of the tail-flick and paw-licking response induced by morphine, DAMGO or DADLE but not β-endorphin or DPDPE. ICI 174864 (5 μg) and ICI 154129 (5 μg) injected i.c.v. selectively antagonized analgesia induced by DPDPE or DADLE but not β-endorphin, morphine or DAMGO injected i.c.v. These results indicate that analgesia induced by morphine and DAMGO is mediated by the stimulation of mu-opioid receptors while analgesia induced by DPDPE is mediated by the stimulation of delta-opioid receptors. DADLE-induced analgesia is mediated by the stimulation of both mu- and delta-opioid receptors. Analgesia induced by β-endorphin is mediated by neither munor delta-opioid receptors.

β-Endorphin-(1–27) antagonizes β-endorphin- but not morphine-, D-PEN2-D-PEN2-enkephalin- and U50, 488H-induced analgesia in mice

β-Endorphin-(1–27), administered intraventricularly has been previously reported to block the analgesia induced by β-endorphin injected intraventricularly. The present study was to determine if the blocking effect of β-endorphin-(1–27) was specific to β-endorphin which stimulates epsilon receptors, but not to other opioids with activity at different opioid receptors. The antagonistic effects of β-endorphin-(1–27) on the analgesia induced by β-endorphin (epsilon-opioid receptor agonist), d-Ala2-NMePhe4-Gly-ol-enkephalin (DAGO) and morphine, (mu-opioid receptor agonists), d-Pen2-d-Pen5-enkephalin (DPDPE) and d-Ala2-d-Leu5-enkephalin(DADLE) (delta-opioid receptor agonists) and U-50, 488H (kappa-opioid receptor agonist) were studied. β-Endorphin-(1–27) injected intraventricularly, at doses which, when injected alone did not produce analgesia, antagonized the analgesia induced by β-endorphin given intraventricularly. However, the analgesia induced by DAGO, morphine, DPDPE, DADLE and U-50,488H given intraventricularly was not antagonized by β-endorphin-(1–27). The data suggest that β-endorphin-(1–27) selectively blocks the analgesia induced by the stimulation of epsilon receptors but not by the stimulation of mu, delta, and kappa receptors. The results support the previously proposed hypothesis that β-endorphin produces its analgesia by stimulating specific epsilon receptors.

Potent antinociceptive effects of TRK-820, a novel κ-opioid receptor agonist

Abstract TRK-820, a new type of 4,5-epoxymorphinan derivative, was investigated in vivo for antinociceptive activities and its selectivity on various opioid receptors in mice. Trk-820 given s.c. or p.o. Was found to be 351- and 796-fold more potent than U50,488H with acetic acid-induced abdominal constriction test. The duration of the antinociceptive effect produced by TRK-820 was longer than that produced by μ-opioid receptor agonist morphine or other κ-opioid receptor agonists. In addition, with four other antinociceptive assays, low temperature hot plate (51 °C), thermal tail flick, mechanical tail pressure and tail pinch tests, TRK-820 was also found to be 68- to 328-fold more potent than U-50488H, and 41- to 349-fold more potent than morphine in producing antinociception, as comparing the weight of the different compound. However, TRK-820 was less active in inhibiting the high temperature (55 ° C) hot plate response. The antinociceptive effects produced by TRK-820 were inhibited by nor-BNI, but not by naloxone or naltrindole (NTI) with the abdominal constriction test, indicating that the antinociception is selectively mediated by the stimulation of κ-, but not μ- or δ- opioid receptors. Co-administration of TRK-820 with morphine slightly enhanced the antinociception induced by morphine in the mouse hot plate test. On the other hand, pentazocine significantly reduced the morphine-induced antinociception. Trk-820 produced sedation at doses, which are much higher than the doses for producing antinociception. These results indicate that the potent antinociception induced by TRK-820 is mediated via the stimulation of κ-, but not μ- or δ-opiod receptors.

Implications of the NR2B subunit-containing NMDA receptor localized in mouse limbic forebrain in ethanol dependence

The present study was designed to further investigate the direct involvement of the NR2B-containing NMDA receptor in ethanol dependence. Using the liquid diet method, mice were chronically treated with skimmed milk containing 5% ethanol for 5 days. After the discontinuation of ethanol, mice revealed tremor, handling-elicited convulsion and death. Treatment with a selective NR2B-containing NMDA receptor antagonist, ifenprodil, significantly suppressed the expression of ethanol withdrawal signs. The protein level of NR2B subunits in the limbic forebrain, but not the cerebral cortex, during chronic ethanol treatment was markedly increased with respect to the levels in control mice. The significant up-regulation of NR2B subunits lasted for at least 9 h after the discontinuation of ethanol and returned to the basal level by 48 h after the withdrawal. These findings suggest that the up-regulation of NR2B subunits during chronic ethanol exposure may be implicated in the initial development of physical dependence on ethanol.