: The rat μ‐opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, shows a desensitization to the inhibitory effect of the μ agonist DAMGO on adenylate cyclase activity ...within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin‐dependent protein kinase II (CaM kinase II) on μ‐opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase II in HEK293 cells. This coexpression led to a faster time course of agonist‐induced desensitization of the μ‐opioid receptor. The increase of desensitization could not be observed with a μ‐opioid receptor mutant (S261A/S266A) that lacks two putative CaM kinase II phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase II in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261A/S266A receptor mutant. These results suggest that phosphorylation of Ser261 and Ser266 by CaM kinase II is involved in the desensitization of the μ‐opioid receptor.
This study investigated whether the metabotropic glutamate receptor ligand (
S)-4C3HPG can reduce brain damage after focal ischemia in rats. Application of 1 μmol of (
S)-4C3HPG ...(intracerebroventricularly) 5 min after occlusion of the middle cerebral artery significantly reduced the infarct size by 72.3% of the saline control.
A novel rat μ opioid receptor (rMOR1B) has been isolated. It shows identity to the recently published sequence of rMOR1 Chen, et al.,
Mol. Pharmacol., 44 (1993) 8–12 up to amino acid 386 and differs ...only in length and amino acid composition at the very carboxy-terminal tail. Both μ opioid receptor isoforms, when stably expressed in CHO-K1 cells, show similar affinities to opioid compounds and are equally effective in the inhibition of forskolin-induced cAMP formation. Reverse transcription polymerase chain reaction (RT-PCR) revealed that rMOR1B displays a similar distribution as rMOR1 in various rat brain areas. Studies measuring the inhibition of adenylate cyclase in cells that had been pre-exposed to the μ opioid agonist DAMGO indicated that rMOR1B is much more resistant to agonist-induced desensitization than rMOR1.
Behavioral experiments demonstrate that stress alters the individual’s attitude towards opiates. In search for the underlying neuronal mechanisms we investigated the influence of stress on ...morphine-induced c-fos expression in the brain, and, vice versa, the influence of morphine application on the brain’s c-fos response to stress. In our experiments, mild stress was induced either by brief immobilization (1 min) or by exposing the rats to a noisy and unfamiliar environment. These kinds of stress, unlike severe stress, did not elicit c-fos expression in the paraventricular nucleus of the hypothalamus. However, c-fos expression was observed in the lateral septum, medial striatum, claustrum and in the cingulate and piriform cortices under these conditions. The stress-induced c-fos induction was markedly decreased by a moderate (10 mg/kg) dose of morphine. On the other hand, morphine alone (50 mg/kg) caused only a weak c-fos expression in naı̈ve animals despite of the rather high dose. If, however, this morphine dose was applied in the presence of a stressful stimulus, a pronounced c-fos expression in the dorsal striatum resulted. This c-fos signal was comparable with the signal seen in morphine-sensitized animals. Thus, distressing conditions seem to alter the brain’s response to morphine at the level of gene expression, and this could be important for initiating voluntary opiate intake.
The decrease in mu-opioid receptor activity after chronic agonist exposure (1 microM D-Ala(2),N-MePhe(4),Gly-ol(5)-enkephalin) is largely due to kinase-mediated phosphorylation of intracellular ...receptor domains. We have recently shown that the substitution of two putative Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) phosphorylation sites, S261 and S266, by alanines in the third intracellular loop of the rat mu-opioid receptor (rMOR1) confers resistance to CaMK II-induced receptor desensitization. In the present study, we show that the injection of active CaMK II in Xenopus laevis oocytes led to the desensitization of S261A but not S266A receptor mutant, indicating that S266 is the primary CaMK II phosphorylation site of the rMOR1. For the corresponding phosphorylation site in the human mu-opioid receptor (hMOR), an allelic variation S268P has been recently identified. After expression in X. laevis oocytes and human embryonic kidney 293 cells, this human S268P receptor and a corresponding rat S266P receptor mutant revealed a loss of CaMK II-induced receptor desensitization and a decreased G protein coupling compared with the wild-type receptors. Our results suggest that serines 266 (rMOR1) and 268 (hMOR) play crucial role in receptor desensitization and signaling and that the allelic variation S268P results in a human receptor type with a weaker but persistent G protein coupling after agonist treatment.
The rat μ opioid receptor gene promoter was cloned and characterized. It has a few features in common with the mouse gene, e.g. the lack of a classical TATA box and the fact that several ...transcriptional start sites are used. The overall homology between the two species is greater than 85%. Functional analysis of the promoter was performed using transient expression of rat μ opioid receptor-reporter gene constructs in the μ opioid receptor expressing cell line SH SY5Y and in non μ opioid receptor expressing cell lines. A promoter region was defined which confers both high basal and TPA and forskolin stimulated reporter gene expression in SH SY5Y cells.
: Prodynorphin, the precursor of the dynorphin opioid peptides, has been shown to play an important role in several aspects of human diseases and complex traits, e.g., drug abuse, epilepsy, and mood ...disorders. The objective of this study was to identify polymorphisms in the 5′ control region of the human prodynorphin gene and to relate these polymorphisms to prodynorphin gene expression. Within the core promoter region, a 68‐bp sequence was found to occur as a polymorphic element, either singular or as tandemly repeated element two, three, or four times. This 68‐bp repeat element contains an AP‐1 transcription factor binding site as demonstrated by electrophoretic mobility shift assay. Reporter gene assays were performed and provided evidence for allele dependent different promoter activity. Dynorphin was found to be involved in many pathophysiological processes so that the described prodynorphin alleles may correlate with the occurrence of several diseases, for example, drug addiction. However, prodynorphin allelic distributions were not significantly different in heroin addicts and control subjects.
Intrathecal administration of octreotide, a stable somatostatin analogue, provides pain relief in patients, and locally applied somatostatin inhibits firing of nociceptive dorsal horn neurons. In the ...present study, we have raised polyclonal antibodies that specifically detect the somatostatin receptor sst2A and used these antisera for immunocytochemical localization of the receptor protein in the rat spinal cord and dorsal root ganglia. In the superficial layers of the dorsal horn, sst2A‐like immunoreactivity (Li) formed a dense network consisting of neuronal perikarya and dendrites which were often closely apposed by, but not co‐contained within, somatostatin‐14‐immunoreactive nerve fibres and terminals. sst2A‐Li was resistant to dorsal rhizotomy and did not colocalize with either substance P or calcitonin gene‐related peptide suggesting that sst2A‐Li was not located to primary afferents, but rather confined to second‐order spinal neurons. The position of sst2A‐Li perikarya and dendrites in the dorsal horn appeared to be similar to those containing μ‐opioid receptor‐Li; however, double labelling experiments revealed no instances of coexistence of these two receptors. sst2A‐Li was also observed in the dorsal root ganglia predominantly targeted to the somatic plasmalemma of medium size neurons distinct from those expressing somatostatin‐14 or δ‐opioid receptors. Thus, the present results not only provide a morphological substrate for spinal octreotide analgesia but also show that somatostatin and opioids are poised to modulate nociceptive transmission by distinct anatomical systems.
The cloning of five members of the somatostatin receptor family, sst
1-sst
5, as well as two isoforms of the somatostatin receptor 2, sst
2A and sst
2B, enabled us to generate specific anti-peptide ...antisera against unique sequences in the carboxyl-terminal tail of each somatostatin receptor subtype. We used these antibodies in multicolor immunofluorescent studies aimed to examine the regional and subcellular distribution of somatostatin receptors in adult rat brain. Several findings are notable: The cloned sst
1 receptor is primarily localized to axons, and therefore most likely functions in a presynaptic manner. The cloned sst
2 receptor isoforms exhibit strikingly different distributions, however, both sst
2A and sst
2B are confined to the plasma membrane of neuronal somata and dendrites, and therefore most likely function in a postsynaptic manner. The cloned sst
3 receptor appears to be excluded from ‘classical’ pre- or postsynaptic sites but is selectively targeted to neuronal cilia. The cloned sst
4 receptor is preferentially distributed to distal dendrites, and therefore most likely functions postsynaptically. The cloned sst
5 receptor was not detectable in the adult rat brain, however, prominent sst
5 expression was found in the pituitary. Furthermore, sst
1-containing axons either co-contained somatostatin or were closely apposed by somatostatin-positive terminals in a regional-specific manner. Neuronal somata and dendrites containing either sst
2A, sst
2B or sst
4 were found to exist in close proximity, although not necessarily synaptically linked, to somatostatin-positive terminals. Together, in the central nervous system the effects of somatostatin are mediated by several different receptor proteins which are distributed with considerable regional overlap. However, there appears to be a high degree of specialization among somatostatin receptor subtypes with regard to their subcellular targeting. This subtype-selective targeting may be the underlying principal of organization that allows somatostatinergic modulation of neuronal activity via both pre- and postsynaptic mechanisms.
Opioid dependence is widely believed to result from neuroadaptations in specific brain regions. However, the precise molecular mechanisms underlying these adaptations are not yet clear. Our aim was ...to explore the role of mitogen‐activated protein kinase (MAPK) in μ opioid receptor signalling in vivo. Using anti‐phospho MAPK antibodies, activated MAPK was detected in cortical neurons (layers II/III), median eminence, amygdaloid and hypothalamic nuclei in untreated animals. Dense nuclear and cytoplasmic staining was observed resulting in full visualization of processes in these cells. Chronic, but not acute, administration of morphine greatly diminished this staining pattern while μ opioid receptor levels and levels of MAPkinase as detected with a phosphorylation state‐independent antibody were unchanged. When opioid withdrawal was precipitated with naloxone a dramatic increase in MAPkinase phosphorylation was observed in somata and fibres of locus coeruleus, solitary tract and hypothalamic neurons. Thus, the differential activation state of MAPK could have important implications for understanding the mechanisms underlying opioid tolerance and dependence.
PDF
