William L. Dewey

μ-opioid receptors: correlation of agonist efficacy for signalling with ability to activate internalization.

We have compared the ability of a number of μ-opioid receptor (MOPr) ligands to activate G proteins with their abilities to induce MOPr phosphorylation, to promote association of arrestin-3 and to cause MOPr internalization. For a model of G protein-coupled receptor (GPCR) activation where all agonists stabilize a single active conformation of the receptor, a close correlation between signaling outputs might be expected. Our results show that overall there is a very good correlation between efficacy for G protein activation and arrestin-3 recruitment, whereas a few agonists, in particular endomorphins 1 and 2, display apparent bias toward arrestin recruitment. The agonist-induced phosphorylation of MOPr at Ser375, considered a key step in MOPr regulation, and agonist-induced internalization of MOPr were each found to correlate well with arrestin-3 recruitment. These data indicate that for the majority of MOPr agonists the ability to induce receptor phosphorylation, arrestin-3 recruitment, and internalization can be predicted from their ability as agonists to activate G proteins. For the prototypic MOPr agonist morphine, its relatively weak ability to induce MOPr internalization can be explained by its low agonist efficacy.

A comparison of some pharmacological actions of morphine and Δ9-tetrahydrocannabinol in the mouse

The effects of morphine and Δ9-tetrahydrocannabinol (THC) on the tail-flick reflex, body temperature, and catecholamine synthesis were examined in the mouse in order to compare their effects in a single species and strain under uniform conditions. Naloxone antagonism of THC and cross-tolerance between morphine and THC were also studied. Both morphine and THC produced antinociception, hypothermia, and increased catecholamine synthesis at 30 min after s.c. injection. Morphine produced greater increases in dopamine synthesis and was a more potent antinociceptive agent, while THC produced greater increases in norepinephrine synthesis and was a more potent hypothermic agent. Naloxone pretreatment (1 mg/kg) partially antagonized the hypothermia and increase in catecholamine synthesis produced by THC. There was also crosstolerance between morphine and THC, but it was asymmetric in that THC-tolerant animals were crosstolerant to only the hypothermic action of morphine and morphine-tolerant animals cross-tolerant to only the antinociceptive action of THC.

Protein kinase inhibitor peptide (PKI): A family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous system and the rest of the body.

Dynorphin-(1–13): Effects in nontolerant and morphine-dependent rhesus monkeys

Abstract Dynorphin-(1–13) (Dyn) substitutes for morphine in morphine-dependent monkeys. At doses which substitute for morphine, this peptide did not produce morphine-like overt effects in nontolerant monkeys. In vivo, Dyn does not behave as a typical κ-agonist. These results are consistent with the suggestion that Dyns role may be that of a regulatory peptide.

Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of µ-opioid receptors in mature brain neurons

Background and purpose:  The ability of an agonist to induce desensitization of the µ‐opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist‐specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high‐efficacy agonist).

Role of protein kinase C and μ-opioid receptor (MOPr) desensitization in tolerance to morphine in rat locus coeruleus neurons

In morphine tolerance a key question that remains to be answered is whether μ‐opioid receptor (MOPr) desensitization contributes to morphine tolerance, and if so by what cellular mechanisms. Here we demonstrate that MOPr desensitization can be observed in single rat brainstem locus coeruleus (LC) neurons following either prolonged (> 4 h) exposure to morphine in vitro or following treatment of animals with morphine in vivo for 3 days. Analysis of receptor function by an operational model indicated that with either treatment morphine could induce a profound degree (70–80%) of loss of receptor function. Ongoing PKC activity in the MOPr‐expressing neurons themselves, primarily by PKCα, was required to maintain morphine‐induced MOPr desensitization, because exposure to PKC inhibitors for only the last 30–50 min of exposure to morphine reduced the MOPr desensitization that was induced both in vitro and in vivo. The presence of morphine was also required for maintenance of desensitization, as washout of morphine for > 2 h reversed MOPr desensitization. MOPr desensitization was homologous, as there was no change in α2‐adrenoceptor or ORL1 receptor function. These results demonstrate that prolonged morphine treatment induces extensive homologous desensitization of MOPrs in mature neurons, that this desensitization has a significant PKC‐dependent component and that this desensitization underlies the maintenance of morphine tolerance.

The spinal cord as a major site for the antinociceptive action of nicotine in the rat

These studies were conducted to localize the antinociceptive action of nicotine within the CNS. Antinociceptive and biodispositional studies were carried out after the injection of [3H]nicotine subcutaneously and intracerebroventricularly into the common carotid and vertebral arteries and into the subarachnoid space. The data indicated that [3H]nicotine was most potent when given into the subarachnoid space than by any of the other route of administration. Further, the disposition studies showed that [3H]nicotine was almost entirely contained in the thoracic and lumbar areas. These results are consistent with the hypothesis that the spinal cord is an important site for antinociception induced by nicotine.

Involvement of phospholipid signal transduction pathways in morphine tolerance in mice.

Opioid tolerance involves an alteration in the activity of intracellular kinases such as cyclic AMP‐dependent protein kinase (PKA). Drugs that inhibit PKA reverse morphine antinociceptive tolerance. The hypothesis was tested that phospholipid pathways are also altered in morphine tolerance. Inhibitors of the phosphatidylinositol and phosphatidylcholine pathways were injected i.c.v. in an attempt to acutely reverse morphine antinociceptive tolerance. Seventy‐two hours after implantation of placebo or 75 mg morphine pellets, mice injected i.c.v. with inhibitor drug were challenged with morphine s.c. for generation of dose‐response curves in the tail‐flick test. Placebo pellet‐implanted mice received doses of inhibitor drug having no effect on morphines potency, in order to test for tolerance reversal in morphine pellet‐implanted mice. Injection of the phosphatidylinositol‐specific phospholipase C inhibitor ET‐18‐OCH3 significantly reversed tolerance, indicating a potential role for inositol 1,4,5‐trisphosphate (IP3) and protein kinase C (PKC) in tolerance. Alternatively, phosphatidylcholine‐specific phospholipase C increases the production of diacylglycerol and activation of PKC, without concomitant production of IP3. D609, an inhibitor of phosphatidylserine‐specific phospholipase C, also reversed tolerance. Heparin is an IP3 receptor antagonist. Injection of low molecular weight heparin also reversed tolerance. PKC was also examined with three structurally dissimilar inhibitors. Bisindolylmaleimide I, Go‐7874, and sangivamycin significantly reversed tolerance. Chronic opioid exposure leads to changes in phospholipid metabolism that have a direct role in maintaining a state of tolerance. Evidence is accumulating that opioid tolerance disrupts the homeostatic balance of several important signal transduction pathways.

Effects of Glucose and Diabetes on Binding of Naloxone and Dihydromorphine to Opiate Receptors in Mouse Brain

The effects of glucose and diabetes on the high-affinity lofentanil-displaceable opiate-receptor binding in mouse brain membranes were studied to determine if the attenuation of opiate actions by hyperglycemia previously observed in our laboratory was due to a modification of receptor affinity or number. With embranes from normal ICR mice, glucose (100–400 mg/dl) caused small but significant concentrationdependent decreases in receptor affinities for [3H]naloxone and [3H]dihydromorphine, both in the absence and presence of 20 mM NaCI, without changing the maximum number of binding sites. Fructose and the nonmetabolizable sugar 3-O-methylglucose had intermedi ate effects on naloxone affinity in the presence of NaCI that were not significantly different from control or from the effect of glucose. Similar results were obtained with brain membranes from streptozocin-induced diabetic mice. The binding affinity for [3H]naloxone in the presence of NaCI was not affected by the induction of diabetes in ICR mice via streptozocin or in spontaneously diabetic (db/db) C57BL/KsJ mice compared with their nondiabetic (m+/m+) litter mates. These results indicate that the previously observed attenuation of opiate effects by glucose may be partly due to a glucose-induced decrease in opiate-receptor affinity. However, the much greater attenuation of morphine by fructose in vivo cannot be explained by this mechanism.

Determination of the role of conventional, novel and atypical PKC isoforms in the expression of morphine tolerance in mice.

Abstract This study comprehensively determines the role of all the major PKC isoforms in the expression morphine tolerance. Pseudosubstrate and receptors for activated C‐kinase (RACK) peptides inhibit only a single PKC isoform, while previously tested chemical PKC inhibitors simultaneously inhibit multiple isoforms making it impossible to determine which PKC isoform mediates morphine tolerance. Tolerance can result in a diminished effect during continued exposure to the same amount of substance. In rodents, morphine pellets provide sustained exposures to morphine leading to the development of tolerance by 72 h. We hypothesized that administration of the PKC isoform inhibitors i.c.v. would reverse tolerance and reinstate antinociception in the tail immersion and hot plate tests from the morphine released solely from the pellet. Inhibitors to PKCα, γ and &egr; (100–625 pmol) dose‐dependently reinstated antinociception in both tests. The PKCβI, βII, δ, &thgr;, &egr;, η and ξ inhibitors were inactive (up to 2500 pmol). In other mice, the degree of morphine tolerance was determined by calculating ED50 and potency‐ratio values following s.c. morphine administration. Morphine s.c. was 5.6‐fold less potent in morphine‐pelleted vs. placebo‐pelleted mice. Co‐administration of s.c. morphine with the inhibitors i.c.v. to either PKCα (625 pmol), γ (100 pmol) or &egr; (400 pmol) completely reversed the tolerance so that s.c. morphine was equally potent in both placebo‐ and morphine‐pelleted mice. The PKCβI, βII, δ, &thgr;, &egr;, η and ξ inhibitors were inactive. Thus, PKCα, γ and &egr; appear to contribute to the expression of morphine tolerance in mice.