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Morphine administered simultaneously to intracerebroventricular (i.c.v.) and intrathecal (i.t.) sites exhibits synergism, with the antinociceptive potency much greater than would be predicted from a simple addition of the potencies of the same dose administered to either site alone. This synergism was quantified in mice using both a fixed dose method, in which the morphine dose at one site was fixed while the AD50 (antinociceptive dose at 50% effectiveness) of morphine at the other site was determined; and a variable dose method, in which different doses of morphine were administered simultaneously to both sites at a fixed ratio, and the AD50 determined and compared to the AD50 at a single site alone. When animals were made tolerant to morphine by implantation of a 75-mg morphine pellet for 3 days, this synergism was eliminated, so that morphine administered simultaneously to i.c.v. and i.t. sites had an additive effect. However, administration of the peptide DynorphinA-(2-17) i.v. simultaneously to the test doses of morphine in morphine-tolerant animals resulted in a partial restoration of synergism. These results suggest that morphine-induced antinociception is highly dependent on an intact integrated central nervous system system and that the initial tolerance development is the result of a disruption of synergism between the central nervous system sites. Morphine tolerance results not from a reduced sensitivity to morphine at discrete central nervous system sites, but rather from a reduced synergistic interaction of morphine at spinal and supraspinal sites. In support of this conclusion, there was no tolerance observed in morphine-pelleted animals to morphine administered to i.c.v. or i.t. sites alone. DynorphinA-(2-17), a nonopioid peptide has previously been shown to enhance the antinociceptive potency of morphine in morphine-tolerant animals, appears to act by restoring this synergism.

Despite the recent cloning of mu, delta and kappa opioid receptors, a role in opioid receptor function for an opioid binding cell adhesion molecule is supported by several lines of evidence, including inhibition of opioid binding by opioid binding cell adhesion molecule antibodies, down-regulation of opioid binding cell adhesion molecule by chronic opioid agonist treatment of cultured NG108-15 cells, and reduction of opioid binding in NG108-15 cells by transfection of opioid binding cell adhesion molecule antisense cDNA. In the present study, we report that chronic in vivo treatment of mice with morphine results in down-regulation of opioid binding cell adhesion molecule immunoreactivity in primary afferent neurons in dorsal root and trigeminal ganglia as well as their axons. This effect was blocked by the opioid antagonist naloxone. Down-regulation of opioid binding cell adhesion molecule immunoreactivity was not observed in other areas of the central nervous system. Taken together, the previous studies which demonstrated the role played by opioid receptors in regulating release of transmitters from primary afferent neurons and the present findings of a specific regulation of opioid binding cell adhesion molecule expression by chronic exposure to morphine, provides evidence from an in vivo perspective which advances the notion that opioid binding cell adhesion molecule plays a role in the action of opioids.

Previously, we demonstrated that the expression of opiate withdrawal and antinociceptive tolerance can be suppressed by dynorphin (dyn) A-(1-13) in morphine-dependent mice. In this study, it was shown that the normal, endogenous dyn, dyn A-(1-17) also possessed this suppressive property. While using the nonopioid dyn analog, [des-Tyr1]dyn A [dyn A-(2-17)] as a negative control, we discovered unexpectedly that this peptide fragment also suppressed naloxone-induced withdrawal and the expression of morphine tolerance in morphine-dependent mice. Thus, an extensive structure activity relationship was studied using 11 peptide fragments. It was determined that the amino acid sequence of dyn A was required for the suppressive activity because dyn B and alpha-neoendorphin both failed to suppress naloxone-precipitated withdrawal jumping. Of the [des-Tyr1]dyn fragments, the minimal amino acid sequence required to suppress naloxone-induced withdrawal was determined to be dyn A-(2-8), containing the sequence G-G-F-L-R-R-I.

We have previously reported that interleukin-1-induced proliferation of thymocytes is accompanied by the appearance of [3H]morphine binding sites on these cells. In the present study, we have characterized these binding sites. They differ from classical opioid receptors in the brain in several ways, including: 1) lack of stereoselectivity; 2) relatively low affinity (Kd = 50 nM) and high capacity (Bmax = 3 pmol/mg of protein); 3) binding is strongly inhibited by Ca++, Mg++, Mn++ and Cl- ions and 4) binding is inhibited by proteinase K or E and by phospholipase A2 but not trypsin treatment of thymocyte membranes. The binding sites, which were found largely on the CD4+ subset of T-cells, also showed a preference for opioid alkaloids over peptides. These [3H]morphine binding sites may mediate a negative feedback effect on interleukin-1-induced proliferation of thymocytes in vivo.

We previously reported that morphine inhibits macrophage-colony stimulating factor (M-CSF)-induced proliferation of mouse bone marrow cells, both in vivo and in vitro, in a dose-dependent manner. We now report that either dynorphin A-(1-13) or dynorphin A-(1-10) amide, though having no effect on proliferation by themselves at concentrations less than 0.1 mM, can block the inhibitory effect of morphine both in vivo and in vitro, in a dose-dependent manner. The opioid antagonist naloxone can also block morphine's inhibitory effect on bone marrow cell proliferation in vivo, but has no effect in vitro. Dynorphin A-(1-13) was also able to block the dramatic reduction of spleen weight observed in animals chronically treated with morphine. Thus dynorphin, which has previously been shown to antagonize morphine analgesia, is also able to antagonize some of the immunosuppressive effects of morphine.

Mouse thymocytes incubated in vitro with increasing concentrations of interleukin-1 (IL-1) in the presence of phytohemagglutinin (PHA) exhibited a dose-dependent increase in cell proliferation, as measured by [3H]thymidine incorporation. Under these conditions, there was a parallel dose-dependent increase in specific [3H]morphine binding, with a maximum increase of approximately 5-fold over basal levels. The binding sites differ from classical opioid receptors in that they are not stereo-selective. Interleukin-2 was ineffective in promoting either cell proliferation or enhanced opioid binding, but the effects of IL-1 could be mimicked by phorbol myristate acetate (PMA), suggesting the involvement of tyrosine phosphorylation. These results indicate that morphine-binding sites on immune cells can be regulated by cytokine activation.

Opioids have been shown to have diverse effects on the immune system, both in vivo and in vitro, but their interactions on immature progenitor cells have been little studied. We have examined the effects of chronic morphine treatment of mice on colony formation by bone marrow cells in vitro. Bone marrow cells from mice implanted with morphine pellets for 72 h showed a 65% decrease in their response to macrophage colony stimulating factor (M-CSF). In contrast, chronic morphine treatment had no effect on the response of bone marrow cells to granulocyte/macrophage colony stimulating factor (GM-CSF). Removal of the morphine pellets from the mice resulted in a time-dependent reversal of the inhibition of macrophage colony formation, and the inhibition was completely blocked by simultaneous administration of naloxone and morphine pellets to the mice. No inhibition of colony formation was observed in bone marrow cells from mice treated with a single acute dose of morphine. Incubation of bone marrow cells from untreated mice for 7 days with in vitro morphine concentrations as low as 25 microM also reduced macrophage colony formation, and the opioid peptide beta-endorphin was even more potent, significantly reducing macrophage colony formation at concentrations as low as 0.25 microM. In agreement with the in vivo effects, neither opioid in vitro had a significant effect on granulocyte/macrophage colony formation. These results suggest that opioids may significantly alter the maturation of immune cells, which could result in potent effects on overall immune competence.

In previous studies, we observed that dynorphin- (1-13), but not dynorphin-(1-9), can significantly inhibit morphine- or beta-endorphin-induced analgesia despite not having any appreciable analgesic activity itself. Dynorphin-(1-13) showed no inhibitory effect on Sandoz FK33824-induced analgesia. In the present study, we examined the effect of dynorphin on morphine-, beta-endorphin-, D-ala2-D-leu5-enkephalin- or Sandoz FK33824-induced analgesia in both naive, morphine-tolerant and morphine-dependent mice. It was found that although dynorphin may inhibit morphine- or beta-endorphin-induced analgesia in naive animals, the peptide is not effective in inhibiting D-ala2-D-leu5-enkephalin- or Sandoz FK33824-induced analgesia. Dynorphin is also effective in blocking spontaneous withdrawal jumping in morphine-dependent animals. It is suggested that dynorphin-(1-13) may play a modulatory role in regulating analgesia due to morphine or beta-endorphin, but not that due to enkephalin. The action of peptides on morphine- or beta-endorphin-induced analgesia in the naive state is different from that of the tolerant state, suggesting that dynorphin may be involved in the development of morphine tolerance and physical dependence.

Interactions between the weakly analgesic enkephalins and morphine on morphine-induced analgesia were studied. Met-enkephalin exhibited morphine analgesia whereas Leu-enkephalin potentiated it. Both Met- and Leu-enkephalin, when tested alone, were not analgesic. The strongly analgesic FK33824 (Sandoz) compound, like Leu-enkephalin, also potentiated morphine analgesia. Tolerance developed to morphine analgesia but not to Met-enkephalin inhibition of morphine analgesia.

Rats were made tolerant to morphine by subcutaneous implantation of morphine alkaloid pellets. Three days after pellet implantation, withdrawal was induced by pellet removal and was assessed 6 h later. Immediately prior to withdrawal assessment, rats were injected with dynorphin A-(1-13) either i.th. (via a catheter), i.c.v. (via a cannula) or i.v. (via the tail vein). When administered i.th. in the dose range 1.25-5 nmol/rat, dynorphin A-(1-13) attenuated withdrawal over the 40 min observation period. Similarly, dynorphin A-(1-13) administered i.v. (37.5-150 nmol/kg) attenuated withdrawal, though only over the first 20 min following administration. Dynorphin A-(1-13) up to 10 nmol/rat had no effect on withdrawal scores. These data indicate that dynorphin acts at spinal sites to suppress withdrawal in morphine-dependent rats and may play a role in tolerance and dependence mechanisms.

Dynorphin-(1-13), but not dynorphin-(1-9) has been shown to have significant effects on opiate and beta-endorphin-induced analgesia despite not having any appreciable analgesic activity itself. Dynorphin had no effect on Sandoz FK33824 compound-induced analgesia.

Pretreatment of mice with a single injection of morphine, beta-endorphin, Leu-enkephalin, FK33824 or [D-Ala2-D-Leu5]enkephalin, for 3 h increased the ability of naloxone to antagonize the analgesic effects of morphine. However, dynorphin-(1-13) can only antagonize morphine, beta-endorphin or Leu-enkephalin induced increased naloxone efficacy but not FK33824 or [D-Ala2-D-Leu2]enkephalin. Dynorphin itself can also increase naloxone efficacy when treated alone. However, this effect can be prevented when animals are pretreated with dynorphin and naloxone together.

Previous studies from our laboratory have shown that the opioid peptide dynorphin-(1-13), although not analgesic when given by itself, can inhibit morphine-induced analgesia in naive mice and potentiate it in morphine tolerant mice. In the present study, we examined the effect of dynorphin-(1-13) with two other dynorphin-like peptides, alpha-neoendorphin and dynorphin-(1-10) amide, on respiration. Our results show that none of the peptides studied had any significant activity on the respiratory rate in mice when given alone. However, in the presence of morphine, dynorphin-(1-13) antagonized the morphine-induced respiratory rate depression in morphine-tolerant animals; alpha-neoendorphin enhanced the morphine-induced respiratory rate depression in naive but had no effect in morphine-tolerant animals and dynorphin-(1-10) amide had no modulatory effect on the morphine-induced respiratory rate depression in either group of animals.

The synthetic beta-endorphin analogs with the omission of the NH2-terminal [Met]enkephalin segment [beta-endorphin-(6-31) and beta-endorphin-(20-31)] are shown to inhibit morphine- or beta-endorphin-induced analgesia in mice by the tail-flick test, whereas the synthetic NH2-terminal pentadecapeptide beta-endorphin-(1-15) has no inhibitory activity. This study raises the possibility that endogenous inhibiting peptides exist in the brain which play a role in the regulation of endorphin actions.