H. Kenneth Kramer

μ and δ-opioid receptor agonists induce mitogen-activated protein kinase (MAPK) activation in the absence of receptor internalization

Agonist-promoted internalization (endocytosis) of G-protein-coupled receptors (GPCRs), including all three opioid receptor types (μ, δ and κ), has been shown to occur via the clathrin endosomal pathway in response to receptor phosphorylation and the actions of the proteins, β-arrestin and dynamin. Many members of the GPCR family stimulate mitogen-activated protein kinases (MAPK or ERK) activity and, in several cases, it appears that MAPK activation is dependent on receptor internalization. We have reinvestigated the question of whether internalization is obligatory for MAPK activation by opioid receptors, using cell lines expressing the cloned μ or δ receptor. Morphine, which is known to activate both μ and δ receptors, does not induce their rapid internalization into clathrin-coated endosomes. However, morphine produced a robust stimulation of MAPK in both cell lines, as demonstrated by the appearance of phosphorylated MAPK. Moreover, pre-exposure of cells to the internalization inhibitors, concanavalin A or hypertonic sucrose, totally blocked DAMGO (μ-selective agonist) and DTLET (δ-selective agonist)-mediated receptor internalization, yet neither treatment affected MAPK phosphorylation induced by these peptides. Our results provide evidence that receptor internalization is not an obligatory requirement for MAPK activation by μ and δ opioid receptors. Hypotheses are presented to explain the seemingly contradictory results obtained from different laboratories.

Role of Protein Kinase C (PKC) in Agonist‐Induced α‐Opioid Receptor Down‐Regulation

Abstract : Phosphorylation of specific amino acid residues is believed to be crucial for the agonist‐induced regulation of several G protein‐coupled receptors. This is especially true for the three types of opioid receptors (μ, δ, and α), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of μ‐ and δ‐opioid receptors and to potentiate agonist‐induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid‐activated PKC contributes to homologous receptor down‐regulation and then expand our focus to include the exploration of the mechanism(s) by which μ‐opioids produce PKC translocation in SH‐SY5Y neuroblastoma cells. [dAla2,N‐Me‐Phe4,Gly‐ol]enkephalin (DAMGO)‐induced PKC translocation follows a time‐dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control‐treated cells in a process we termed “reverse translocation.” The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L‐type calcium channel antagonist nimodipine attenuated opioid‐mediated effects on PKC and μ‐receptor down‐regulation, suggesting that this is a process partially regulated by Ca2+‐dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+‐sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down‐regulation. In addition to expressing conventional (PKC‐α) and novel (PKC‐ε) isoforms, SH‐SY5Y cells also contain a DAG‐and Ca2+‐independent, atypical PKC isozyme (PKC‐ξ), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC‐ξ is similarly sensitive to activation by μ‐opioids. PKC‐ξ translocates from the cytosol to the membrane with kinetics similar to those of PKC‐α and ε in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by μ‐opioid agonists is involved in the processes that result in μ‐receptor down‐regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.

Tyrosine phosphorylation of the δ-opioid receptor: Evidence for its role in mitogen-activated protein kinase activation and receptor internalization

The internalization of G-protein-coupled receptors (GPCRs), including the delta opioid receptor (delta-OR), has been shown to involve the phosphorylation of serine and threonine residues. However, recent studies suggest that these residues may not be the only ones phosphorylated in response to prolonged opioid exposure. Tyrosines also appear important for delta-OR signalling, but it remains unclear whether they undergo phosphorylation. We examined whether the delta-OR, stably expressed in Chinese hamster ovary (CHO-K1) cells, was tyrosine-phosphorylated during prolonged agonist treatment. The epitope-tagged delta-OR was purified by immunoprecipitation, and the presence of phosphorylated tyrosines was detected using anti-phosphotyrosine antibodies. Tyrosine residues in the delta-OR were phosphorylated after exposure to the high-affinity agonist [d-Thr(2)]-Leu-enkephalin-Thr (DTLET) in a time- and concentration-dependent manner. Tyrosine phosphorylation of the delta-OR appeared to require the actions of a Src-like protein tyrosine kinase, since the Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-[3,4-d]-pyrimidine (PP1) attenuated this response. PP1 also attenuated the DTLET-mediated activation of mitogen-activated protein kinase, as well as rapid delta-OR internalization, but not receptor down-regulation. Finally, only opioid agonists that induce receptor internalization via the clathrin-dependent endosomal pathway stimulated significant tyrosine phosphorylation of the delta-OR protein. Evidence is presented that the delta-OR is tyrosine-phosphorylated, and we suggest how this may have an active role in opioid receptor signalling and regulation.

Delta Opioid activation of the Mitogen-activated protein kinase cascade does not require transphosphorylation of Receptor Tyrosine Kinases

BackgroundIn this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned δ-opioid receptor (δ-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive.ResultsOur results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed δ-ORs are stimulated by the δ-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the δ-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In δ-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation.ConclusionTaken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated δ-OR, itself, is likely to act as its own signalling scaffold.

Emerging evidence for a central epinephrine-innervated alpha 1-adrenergic system that regulates behavioral activation and is impaired in depression.

Currently, most basic and clinical research on depression is focused on either central serotonergic, noradrenergic, or dopaminergic neurotransmission as affected by various etiological and predisposing factors. Recent evidence suggests that there is another system that consists of a subset of brain α1B-adrenoceptors innervated primarily by brain epinephrine (EPI) that potentially modulates the above three monoamine systems in parallel and plays a critical role in depression. The present review covers the evidence for this system and includes findings that brain α1-adrenoceptors are instrumental in behavioral activation, are located near the major monoamine cell groups or target areas, receive EPI as their neurotransmitter, are impaired or inhibited in depressed patients or after stress in animal models, and are restored by a number of antidepressants. This ‘EPI-α1 system’ may therefore represent a new target system for this disorder.

Activation of Protein Kinase C (PKC) by 3,4-Methylenedioxymethamphetamine (MDMA) Occurs Through the Stimulation of Serotonin Receptors and Transporter

This report further characterizes the intermediate metabolic effects of the psychotropic amphetamine derivative, 3,4-methylenedioxymethamphetamine (MDMA or “ecstasy”), on the activity of second messenger-dependent kinases. Previous work has demonstrated that two injections of MDMA (20 mg/kg) elicits a prolonged translocation of the calcium and phospholipid-dependent enzyme, protein kinase C (PKC) in rats. However, because MDMA has actions at the 5-HT transporter and 5-HT2A/2C receptors, our experiments were directed at uncovering which of these many sites may be involved in this second messenger-dependent response. A single injection of MDMA produced a time- and dose-dependent increase in the density of cortical and hippocampal PKC (as measured by 3H-phorbol 12,13-dibutyrate (PDBu)) binding sites. MDMA-mediated PKC translocation was long-lasting and remained above control (saline-treated rats) for up to 24 h after injection. This effect was mimicked by another substituted amphetamine, p-chloroamphetamine (pCA), but with a temporal-response curve that was to the left of MDMA’s. However, pure uptake inhibitors like fluoxetine, cocaine, and the selective 5-HT2A/2C agonist, DOB, were unable to produce a long-lasting translocation of PKC binding sites in rat cortex. Fluoxetine, a selective serotonin uptake inhibitor (SSRI) and ketanserin, a 5-HT2A antagonist, attenuated PKC translocation by MDMA with differing efficacies; however, both compounds completely prevented the loss of 5-HT uptake sties after multiple doses of MDMA. These results suggest that MDMA increases PKC translocation by two interrelated mechanisms that involve 5-HT2A/2C receptors and the 5-HT transporter. This pathway appears to include: (1) the drug binding to the 5-HT transporter, (2) the release of cytosolic 5-HT stores into the extracellular space, and (3) the activation of post-synaptic 5-HT2A/2C receptors linked to G-protein-mediated phospholipid hydrolysis.

Characterization of the translocation of protein kinase C (PKC) by 3,4-methylenedioxymethamphetamine (MDMA/Ecstasy) in synaptosomes: Evidence for a presynaptic localization involving the serotonin transporter (SERT)

3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) is a substituted amphetamine whose acute and long-term effects on the serotonin system are dependent on an interaction with the 5-HT uptake transporter (SERT). Although much of the work dedicated to the study of this compound has focused on its ability to release monoamines, this drug has many important metabolic consequences on neurons and glial cells. The identification of these physiological responses will help to bridge the gap that exists in the information between the acute and neurotoxic effects of amphetamines. Substituted amphetamines have the ability to produce a long-term translocation of protein kinase C (PKC) in vivo, and this action may be crucial to the development of serotonergic neurotoxicity. Our earlier results suggested that PKC activation occurred through pre- and postsynaptic mechanisms. Because the primary site of action of these drugs is the 5-HT transporter, we now expand on our previous results and attempt to characterize MDMAs ability to translocate PKC within cortical 5-HT nerve terminals. In synaptosomes, MDMA produced a concentration-dependent increase in membrane-bound PKC (as measured by 3H-phorbol 12, 13 dibutyrate, 3H-PDBu) bindings sites. This response was abolished by cotreatment with the specific serotonin reuptake inhibitor (SSRI), fluoxetine, but not by the 5-HT2A/2C antagonist, ketanserin. In contrast, full agonists to 5-HT1A and 5-HT2 receptors did not produce significant PKC translocation. MDMA-mediated PKC translocation also requires the presence of extracellular calcium ions. Using assay conditions where extracellular calcium was absent prevented the in vitro activation of PKC by MDMA. Prolonged PKC translocation has been hypothesized to contribute to the calcium-dependent neurotoxicity produced by substituted amphetamines. In addition, many physiological processes within 5-HT nerve terminals, including 5-HT reuptake and vesicular serotonin release, are susceptible to modification by PKC-dependent protein phosphorylation. Our results suggest that prolonged activation of PKC within the 5-HT nerve terminal may contribute to lasting changes in the homeostatic function of 5-HT neurons, leading to the degeneration of specific cellular elements after repeated MDMA exposure.

Specificity versus Redundancy of Melanocortins in Nerve Regeneration

The results of the present study demonstrate that administration of the ACTH-(4-9) analogue Org 2766 acutely enhances behavioral, morphological, and biochemical recovery after nigrostriatal destruction. Animals treated with Org 2766 (10 micrograms/kg every 24 hr) demonstrated an acceleration of denervation supersensitivity and a significantly decreased ipsilateral rotational response, as compared to their saline counterparts. Upon evaluation of the mesolimbic DA system using open field behavior, peptide-treated rats demonstrated a compensatory response in their rearing behavior. Furthermore, tyrosine hydroxylase immunocytochemical analysis indicated an enhanced staining in the Org 2766-treated groups. This evaluation was confirmed and quantified using specific high-affinity dopamine uptake. The brains of animals treated with Org 2766 maintained higher uptake levels, suggesting a greater fiber density than the saline-treated animals. Although recovery via reinnervation is very unlikely in this short period of time, improved recovery may be the result of a protective effect of Org 2766 after administration of 6-OHDA into the substantia nigra. Thus, it appears that Org 2766 provides the rapid effects in this system, by both accelerating some compensatory mechanisms necessary for functional recovery and promoting cell survival by providing neuronal protection. However, it does not appear that this protection is due to NMDA receptor manipulation. Org 2766 neither mimicked the NMDA antagonist MK-801 behaviorally nor biochemically in binding displacement studies. Interestingly, other studies have suggested that only the full ACTH molecule, and fragments larger than ACTH-(1-17), demonstrated binding activity at micromolar concentrations, whereas the shorter, noncorticotropic fragments were either less active or inactive (Table 2). As for ACTH-(4-10) immunoreactivity, it appears that this neurotrophic fragment of ACTH reappears in adults following injury to the nigrostriatal system. In addition, the systemically administered ACTH-(4-9) analogue, Org 2766, seems to be gaining access to the CNS, but is only effective in the injured system. Therefore, based on the immunocytochemical localization of the ACTH-(4-10) fragment in neonatal brains and in the injured adult rat CNS, the interesting possibility may be raised that endogenous ACTH peptides appear during both ontogeny and regeneration. These studies demonstrate once again that biological responses to the family of ACTH/MSH peptides depend on the specific peptide fragment administered, its dosage, and the timing of the administration. Consequently, since early intervention is of vital importance in CNS recovery processes, synergistic administration of ACTH fragments and other neurotrophic agents may offer a viable approach with which to combat degeneration in the CNS.

The effect of nicotine on catecholaminergic storage vesicles

The present study examined the action of nicotine on the accumulation of [3H]dopamine into synaptic vesicles prepared from mouse cerebral cortex or bovine striatum. Nicotine was shown to be a weak inhibitor of [3H]dopamine accumulation, with an IC50 of approximately 0.2-0.4 mM. In addition, repeated nicotine administration (1.2 mg (-)-nicotine di-(+)tartrate/kg s.c., twice daily for 10 days) in vivo in BALB/cBy male mice did not alter the potency of reserpine in inhibiting [3H]dopamine accumulation into synaptic vesicles, nor did it change the slight shift induced by nicotine in the potency of reserpine in inhibiting [3H]dopamine accumulation. The present results show that nicotine is an inhibitor of vesicular dopamine accumulation at high concentrations.