Functional selectivity and time-dependence of µ-opioid receptor desensitization at nerve terminals in the mouse ventral tegmental area.

BACKGROUND AND PURPOSE: The majority of studies examining desensitization of the µ-opioid receptor (MOR) have examined those located at cell bodies. However, MORs are extensively expressed at nerve terminals throughout the mammalian nervous system. This study is designed to investigate agonist-induced MOR desensitization at nerve terminals in the mouse ventral tegmental area (VTA). EXPERIMENTAL APPROACH: MOR function was measured in mature mouse brain slices containing the VTA using whole-cell patch-clamp electrophysiology. Presynaptic MOR function was isolated from postsynaptic function and the functional selectivity, time-dependence and mechanisms of agonist-induced MOR desensitization were examined. KEY RESULTS: MORs located at GABAergic nerve terminals in the VTA were completely resistant to rapid desensitization induced by the high-efficacy agonists DAMGO and Met-enkephalin. MORs located postsynaptically on GABAergic cell bodies readily underwent rapid desensitization in response to DAMGO. However, after prolonged (>7 h) treatment with Met-enkephalin, profound homologous MOR desensitization was observed. Morphine could induce rapid MOR desensitization at nerve terminals when PKC was activated. CONCLUSIONS AND IMPLICATIONS: Agonist-induced MOR desensitization in GABAergic neurons in the VTA is compartment-selective as well as agonist-selective. When MORs are located at cell bodies, higher-efficacy agonists induce greater levels of rapid desensitization than lower-efficacy agonists. However, the converse is true at nerve terminals where agonists that induce MOR desensitization via PKC are capable of rapid agonist-induced desensitization while higher-efficacy agonists are not. MOR desensitization induced by higher-efficacy agonists at nerve terminals only takes place after prolonged receptor activation. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Interleukin-15 administration increases graft-versus-tumor activity in recipients of haploidentical hematopoietic SCT.

Utilizing a clinically relevant haploidentical (HI) murine transplant model, lethally irradiated B6D2F1 (H2K(b/d)) mice were transplanted with T cell-depleted (TCD) BM from B6CBAF1 (H2K(b/k)) mice. We found that administration of IL-15 significantly increases the numbers of CD8+ T and natural killer (NK) cells in spleen and BM after transplantion without GVHD. Graft-versus-tumor (GVT) potency of the graft was evaluated upon tumor challenge using P815 tumor cells (H2(d)). IL-15 administration without T-cell infusion did not result in any survival improvement. However, IL-15 in combination with very low-dose T-cell infusion (1 × 10(4)) significantly increased GVT activity and improved survival in recipients of HI hematopoietic SCT (HSCT). This effect was observed when IL-15 was given at a later time point, rather than immediately following transplantation. IL-15 administration also specifically increased slow-proliferative CD8+ T-cell proliferation and IFN-γ secretion in CD8+ T cells in recipients of CFSE (carboxyfluorescein succinimidyl ester)-labeled HI T-cell infusion, whereas there was no effect on CD4+ T-cell proliferation, suggesting the critical effect of IL-15 on CD8+ T-cell homeostasis in HI host. We conclude that IL-15 can be used for enhancing antileukemia effect of HI-HSCT, which requires presence of donor-derived T cells.

Reversal of morphine analgesic tolerance by ethanol in the mouse.

The chronic use of opioids in humans, accompanied by the development of tolerance, is a dangerous phenomenon in its own right. However, chronic opioid use is often made more dangerous by the coconsumption of other substances. It has been observed that the blood level of opioids in postmortem analyses of addicts, who consumed ethanol along with the opioid, was much less than that observed in individuals who died from opioids alone. This relationship between ethanol and opioids led us to investigate the hypothesis that ethanol alters tolerance to opioids. In the present study, we report that ethanol significantly and dose-dependently reduced the antinociceptive tolerance produced by morphine and the cross-tolerance between [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and morphine in the mouse tail-flick test. The reversal of morphine tolerance was partially blocked by both the gamma receptor blocker bicuculline and by the γ-aminobutyric acid (GABA)(B) receptor blocker phaclofen and the administration of both inhibitors completely reversed the effects of ethanol on morphine tolerance. Diazepam, like ethanol, decreased morphine tolerance. However, this inhibition was reversed by the GABA(A) antagonist bicuculline but not by the GABA(B) antagonist phaclofen. These findings have important implications for individuals who abuse opioids and ethanol as well as suggest a mechanism to reduce the amount of opioid needed in chronic pain treatment.

Haploidentical hematopoietic SCT increases graft-versus-tumor effect against renal cell carcinoma.

Allogeneic hematopoietic SCT (HSCT) has been shown to be an effective treatment option for advanced renal cell cancer (RCC). However, tumor resistance/relapse remains as the main post transplant issue. Therefore, enhancing graft-versus-tumor (GVT) activity without increasing GVHD is critical for improving the outcome of HSCT. We explored the GVT effect of haploidentical-SCT (haplo-SCT) against RCC in murine models. Lethally irradiated CB6F1 (H2K(b/d)) recipients were transplanted with T-cell-depleted BM cells from B6CBAF1 (H2K(b/k)) mice. Haplo-SCT combined with a low-dose haploidentical (HI) T-cell infusion (1 × 10(5)) successfully provided GVT activity without incurring GVHD. This effect elicited murine RCC growth control and consequently displayed a comparative survival advantage of haplo-SCT recipients when compared with MHC-matched (B6D2F1CB6F1) and parent-F1 (B6CB6F1) transplant recipients. Recipients of haplo-SCT had an increase in donor-derived splenic T-cell numbers, T-cell proliferation and IFN-γ-secreting donor-derived T-cells, a critical aspect for anti-tumor activity. The splenocytes from B6CBAF1 mice had a higher cytotoxicity against RENCA cells than the splenocytes from B6 and B6D2F1 donors after tumor challenge. These findings suggest that haplo-SCT might be an innovative immunotherapeutic platform for solid tumors, particularly for renal cell carcinoma.

Involvement of PKC alpha and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons.

BACKGROUND AND PURPOSE: The ability of an agonist to induce desensitization of the mu-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). EXPERIMENTAL APPROACH: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). KEY RESULTS: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. CONCLUSIONS AND IMPLICATIONS: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

Role of protein kinase C and mu-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 mu-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 PKCalpha, 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 alpha(2)-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.

Agonist-selective mechanisms of GPCR desensitization.

The widely accepted model of G protein-coupled receptor (GPCR) regulation describes a system where the agonist-activated receptors couple to G proteins to induce a cellular response, and are subsequently phosphorylated by a family of kinases called the G protein-coupled receptor kinases (GRKs). The GRK-phosphorylated receptor then acts as a substrate for the binding of a family of proteins called arrestins, which uncouple the receptor and G protein so desensitizing the agonist-induced response. Other kinases, principally the second messenger-dependent protein kinases, are also known to play a role in the desensitization of many GPCR responses. It is now clear that there are subtle and complex interactions between GRKs and second messenger-dependent protein kinases in the regulation of GPCR function. Functional selectivity describes the ability of agonists to stabilize different active conformations of the same GPCR. With regard to desensitization, distinct agonist-activated conformations of a GPCR could undergo different molecular mechanisms of desensitization. An example of this is the mu opioid receptor (MOPr), where the agonists morphine and [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) induce desensitization of the MOPr by different mechanisms, largely protein kinase C (PKC)- or GRK-dependent, respectively. This can be best explained by supposing that these two agonists stabilize distinct conformations of the MOPr, which are nevertheless able to couple to the relevant G-proteins and produce similar responses, yet are sufficiently different to trigger different regulatory processes. There is evidence that other GPCRs also undergo agonist-selective desensitization, but the full therapeutic consequences of this phenomenon await further detailed study.

Neurokinin-1 receptors in the rat nucleus tractus solitarius: pre- and postsynaptic modulation of glutamate and GABA release.

Neurokinins such as substance P and neurokinin A have long been thought to act as neurotransmitters or modulators in the nucleus tractus solitarius. However, the role and location of the receptors for these peptides have remained unclear. We examined the consequences of activation of the neurokinin-1 (NK1) receptor subtype in the rat nucleus tractus solitarius using whole-cell patch clamp recordings in brain slices. Application of delta-Ala-Phe-Phe-Pro-MeLeu-D-Pro[spiro-gamma-lactam]-Leu-Trp-NH2 (a specific NK1 agonist) or neurokinin A resulted in depolarization, evident as a slow inward current, mediated by direct postsynaptic NK1 receptor activation. The effect was conserved in the presence of tetrodotoxin, and protein kinase C-dependent since it was blocked by 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide, a specific protein kinase C inhibitor. In addition, an increase in the frequency and amplitude of spontaneous excitatory postsynaptic currents was observed, reflecting increased glutamate release induced by NK1 receptor activation. This effect was abolished by tetrodotoxin, suggesting that it resulted from increased firing in afferent neurons, subsequent to somatodendritic excitation via NK1 receptors. Furthermore, spontaneous inhibitory postsynaptic currents were increased in frequency and amplitude showing that GABA release was promoted by NK1 receptor activation. However, amplitude of miniature inhibitory postsynaptic currents was unaltered by NK1 receptor activation, but the increase in frequency persisted. These findings suggest that NK1 receptors are located on presynaptic terminals as well as at somatodendritic sites of GABAergic neurons. The increase in GABA release was also shown to be protein kinase C-dependent. The data presented here show NK1 receptors in the rat nucleus tractus solitarius are present both excitatory and inhibitory neurons. Activation of these receptors can result in increases in release of both GABA and glutamate, suggesting a crucial modulatory role for NK1 receptors in the rat nucleus tractus solitarius.

Alterations in mesolimbic dopamine function during the abstinence period following chronic ethanol consumption.

Previous work demonstrated that the locomotor stimulant actions of amphetamine, cocaine and nicotine were increased when these drugs were given during the abstinence phase after chronic ethanol consumption. These changes were seen at 6 days and at 2 months after cessation of alcohol. The present study examined neuronal alterations which might be related to these changes in behaviour. Markedly reduced spontaneous firing rates of dopaminergic cells in the ventral tegmental area (VTA) in midbrain slices were seen 6 days into the abstinence period after cessation of chronic ethanol consumption, but by 2 months the firing rates had returned to control values. Increased affinity of striatal receptors for the D1-like receptor ligand 3H-SCH23390, but no change in the receptor density, was found both at the 6 day and the 2 month intervals. The binding properties of striatal D2-like receptors, of D1-like and D2-like receptors in the frontal cerebral cortex, and the release of tritiated dopamine from slices of striatum or frontal cerebral cortex, were unchanged at 6 days and 2 months. It is suggested that the decreased neuronal firing leads to a persistent increase in sensitivity of D1-like receptors and that these changes could explain the increased effects of the other drugs of abuse.

Prolonged changes in neurochemistry of dopamine neurones after chronic ethanol consumption.

The effects of 3 weeks of chronic ethanol consumption in mice on brain concentrations and turnover of monamine transmitters was examined. The measurements were made at 24 h, 6 days and 2 months after cessation of the ethanol intake to examine changes that might be relevant to relapse drinking. Increases in noradrenaline and dopamine concentrations, and decreases in the ratios of dopamine metabolites to dopamine, were seen in ventral tegmental tissue at 24 h after alcohol consumption. Increased noradrenaline was also evident at the 6-day interval, but no other changes were seen at this time. At the 2-month interval, the ventral tegmentum from ethanol-treated animals showed decreases in metabolite/dopamine ratios. No changes were seen in 5-hydroxytryptamine or its metabolite. In striatal tissue, none of these changes were seen, but at 24 h decreases occurred in the content of dopamine and its metabolites and a decrease in 5-hydroxyindoleacetic acid. The results indicate changes occur in monoamine turnover in the VTA as long as 2 months after cessation of chronic ethanol consumption; such changes may be related to the prolonged nature of alcohol dependence.

Cationic oligonucleotides can mediate specific inhibition of gene expression in Xenopus oocytes.

Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selective inhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). When compared with unmodified oligonucleotides of identical base composition, DEED-modified oligonucleotides were better able to form DNA triplexes under conditions that approximate the pH, magnesium and potassium levels found in vivo . Here we report the ability of DEED-modified oligonucleotides to inhibit the expression of plasmid DNA injected into Xenopus oocytes. Inhibition is specific to plasmids containing a triplex formation target and sensitive to sequence alteration in the triplex forming target site. Inhibition of gene expression was nearly complete when oligonucleotide and plasmid were mixed together prior to injection. Inhibition was partial when oligonucleotide was injected first and not evident when plasmid was injected and allowed to form chromatin prior to oligonucleotide injection. Thus, access to DNA is a determining factor in effective triplex inhibition of gene expression.

Chronic ethanol administration alters activity in ventral tegmental area neurons after cessation of withdrawal hyperexcitability.

The present study investigated the activity of neurons in the mesolimbic dopamine system after the end of the acute phase of the behavioural signs of ethanol withdrawal in mice. This was designed to provide a comparison with earlier behavioural studies, in which greater development of sensitisation to amphetamine and cocaine, but no change in the initial effects of these compounds, or in the behaviour in the absence of drug treatment, was seen when repeated injection of these psychostimulants were given after chronic ethanol consumption. In the present study, single unit recordings were made from dopamine-sensitive neurons in the ventral tegmental area in perfused midbrain slices prepared 24 h after cessation of chronic ethanol consumption. Profound decreases in firing of the ventral tegmental area (VTA) neurons were seen in slices prepared after the ethanol treatment. Firing rates increased after application of N-methyl-dl-aspartate, but still remained lower and more variable after the ethanol treatment. Application of dopamine or amphetamine, following stimulation of firing with a low concentration of N-methyl-dl-aspartate, also resulted in lower firing rates in slices from ethanol-treated mice. No changes were seen in release of tritiated dopamine, in response to applied KCl or amphetamine, from slices of striatum or cerebral cortex, prepared 24 h after cessation of the chronic ethanol consumption, compared with control values. The results demonstrate that very substantial decreases in firing rate, and in the number of active cells, occur in VTA neurons at a time when withdrawal hyperexcitability was no longer apparent and overt changes in behaviour were not seen.

Comparison of the effects of drugs on hyperexcitability induced in hippocampal slices by withdrawal from chronic ethanol consumption.

1 The effects of drugs, previously demonstrated to have a range of effects on the behavioural signs of ethanol withdrawal hyperexcitability, were examined in area CA1 in isolated hippocampal slices prepared after withdrawal from chronic ethanol in vivo. 2 The decreases seen after the ethanol treatment in the thresholds for production of single and multiple population spikes were prevented when the dihydropyridine calcium channel antagonist, isradipine, was included in the perfusion medium at 4 microM. 3 Another dihydropyridine, felodipine, which had no activity against withdrawal signs in vivo, did not affect the changes in field potentials, at concentrations up to 10 microM. 4 Diltiazem, which increased withdrawal hyperexcitability in vivo, had no effect on the withdrawal changes in field potentials at 30 microM; higher concentrations affected the control slices. 5 The novel anticonvulsant, gabapentin, at 1 microM but not at 100 nM, significantly decreased the signs of withdrawal hyperexcitability in the hippocampal slices. When the CCKB antagonist, CI988, was added to the bathing medium, at 1 microM, there were small, but significant decreases in the withdrawal hyperexcitability. 6 The results showed that the actions of these drugs on the changes in the field potentials in isolated hippocampal slices were very similar to their previously demonstrated effects on the convulsive signs of ethanol withdrawal in vivo, but differences were seen in the corresponding comparison with anxiolytic actions in vivo.

Two related localized mRNAs from Xenopus laevis encode ubiquitin-like fusion proteins.

The uneven distribution of maternal mRNAs in unfertilized eggs and the unequal inheritance of these molecules by dividing blastomeres may be one mechanism for determining cell fate during embryogenesis. Complementary DNA (cDNA) clones corresponding to maternal mRNAs localized to specific regions of the Xenopus laevis egg have been previously identified and cloned [Rebagliati et al., Cell 42(1985) 769-777]. The maternal mRNA, An1, was originally identified as being localized to the animal hemisphere of X. laevis eggs and early embryos. We describe here the two proteins encoded by two An1 mRNA isoforms which we designate An1a and An1b. These mRNAs are both approximately 3.0 kb long and are concentrated in the animal hemisphere of unfertilized eggs. The predicted amino acid (aa) sequences encoded by An1a and An1b correspond to 76.9 and 78.6 kDa, respectively, and are 88% identical. Both proteins contain a single N-terminal ubiquitin (Ub)-like domain (50% identical to X. laevis Ub) and a putative Zn(2+)-binding region near the C terminus. Unlike Ub polyproteins and most Ub fusion proteins, the N-terminal Ub-like domain found in the An1 proteins does not undergo proteolytic processing. In contrast to earlier studies showing that the An1 mRNA represents a strictly maternal transcript, we report that both related An1 transcripts are found in later embryonic stages and in all adult tissues tested.

Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction.

The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

High pressure and intravenous steroid anesthesia in rats.

Anesthesia produced by the intravenous steroid agent, Althesin, was studied in Sprague-Dawley rats with and without high pressure of helium gas up to 100 atmospheres absolute (ATA). There were no cumulative or adaptive changes in Althesin requirement at normal pressures over 6-h periods. However, the apparent potency of the agent was reduced by 43% by the addition of 68 ATA helium. Subanesthetic doses of Althesin protected against the onset of convulsions and coarse tremors associated with the high pressure neurological syndrome. It is concluded that the steroid anesthetics may have a place in human diving technology and that the mechanisms associated with the anesthetic-pressure interactions are consistent with the critical volume or lipid bilayer fluidity hypothesis.

Reconstruction of the cardiac valves with autologous tissue.

A rather long and extensive experience with tissue reconstruction in patients with mitral valve disease, and a much less extensive one with oartic lesions, has convinced us of the superiority of the presented techniques of reconstruction, and of the preferability of fascia lata over all other tissues so far tried for this purpose. Despite an early increment of shrinkage of the order of about 30% of each linear measurement, late studies of fascia lata removed from reconstructed valves after several years (over five) indicate no loss of cellularity and no measurable loss of tissue strength or flexibility. Late calcification was not observed in any of our baboons, although it appears to be a consistent development in dogs. It has been seen in only one patient (after four years) to date. It is now believed that we can offer prolonged clinical benefit approaching actural "cure" to many of the younger patients who otherwise would have no recourse but to prosthetic palliation. It is true that recently Willen, Dubiel and Johansson (50, 51), Gersbach and Wegmann (52), and Senning and Rothlin (53) have demonstrated that repetitive deposits of fibrin upon the surfaces of fascia lata implanted within the cardiovascular chambers lead to progressive encapsulation with organizing connective tissue (scar). At some time period following surgery, closer to 10 years than to 5, degeneration of the fascia takes place, presumably due to "strangulation" by the organized exudate which interrupts the "normal" mechanism of its nutrition which is based upon diffusion from the flowing blood. The recent contributions of Sullivan, Harken and Gorlin (54), Weily and Genton (55), and Harker and Slicter (56) to our understanding of the role of the platelets in initiating such fibrinous deposition now provide us with a way to prevent such late degeneration of valves made of fascia lata. The regular administration of platelet dispersing agents (aspirin, Persantin, or inderol) in ordinary therapeutic dosage would seem to be completely protective. Undoubtedly, anticoagulant therapy would be equally effective. However, the permanent maintenance of a proper level of "anti-coagulation" such as is usually deemed necessary following implantation of a prosthetic heart valve is a heavy psychological and biological burden for a patient to bear. Many such individuals live precariously between the risks of thromboembolism and the risks of hemorrhage. Thromboembolism really only represents a farther point along the spectrum of the readiness of fibrin accumulation following initial platelet aggregation and deposition. Since frank thromboembolism appears "never" to follow intracardiac implantation of fascia lata, it would seem that platelet dispersive therapy sould suffice in such cases.