Richard D. Howells

Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors.

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged δ and μ receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [35S]methionine metabolic labeling indicated that the turnover rate of δ receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional Gi and Go proteins by pertussis toxin-attenuated down-regulation of the μ opioid receptor, while down-regulation of the δ opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on μ and δ opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced μ and δ receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state μ and δ opioid receptor levels. Immunoprecipitation of μ and δ opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.

Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior

The ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM) contains many estrogen-concentrating neurons which mediate estrogen facilitation of reproductive behavior. Previous studies have shown that estrogen treatment increases proenkephalin (PE) gene expression in neurons of the VL-VM in ovariectomized female rats, and that enkephalin peptides may stimulate lordosis behavior. To determine whether there is a sex difference in steroid hormone regulation of PE gene expression we have examined the effects of estrogen and testosterone on PE mRNA levels in male rats. Slot blot hybridization analysis of RNA isolated from the ventromedial hypothalamus indicated that estrogen treatment increased PE mRNA levels in the VL-VM of ovariectomized female rats (2.2-fold), but had no measurable effect on PE mRNA levels in gonadectomized males. Testosterone treatment of gonadectomized males also had no effect on PE gene expression. To determine whether the sex difference in estrogen-inducibility of PE gene expression is due to the developmental effects of gonadal steroids, we have investigated the effect of estrogen on PE mRNA levels in the VL-VM of neonatally androgenized female rats. Unlike the genetic male, the androgenized females responded to estrogen treatment with a female-typical increase in PE mRNA levels (1.7-fold). Further, although the androgenized rats clearly exhibited signs of defeminization, they did exhibit estrogen-facilitated lordosis behavior when tested with manual stimulation. The PE mRNA induction in estrogen-treated androgenized rats correlated well with the lordosis scores obtained by manual stimulation testing. These results indicate that estrogen regulation of PE gene expression in the VL-VM is sexually differentiated and support the hypothesis that the enkephalinergic neurons of the VL-VM are involved in the regulation of female reproductive behavior.

Structure and regulation of opioid receptors.

Significant advances have been made in understanding the structure, function, and regulation of opioid receptors and endogenous opioid peptides since their discovery approximately 25 years ago. This review summarizes recent studies aimed at identifying key amino acids that confer ligand selectivity to the opioid receptors and that are critical constituents of the ligand binding sites. A molecular model of the delta receptor based on the crystal structure of rhodopsin is presented. Agonist-induced down regulation of opioid receptors is discussed, highlighting recent evidence for the involvement of the ubiquitin/proteasome system in this process.

μ Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity

The importance of the amino-terminal domain of the mu opioid receptor (MOR) as a component of the high affinity ligand-binding pocket was evaluated. A deletion mutant lacking 64 amino acids from the amino-terminus of MOR (DeltaN64) was constructed and expressed in HEK 293 cells. The affinities of bremazocine and cyclazocine were similar for the truncated and full-length MORs. Affinities of the mu receptor antagonist, naloxone, and the mu receptor agonist, morphine, were decreased 3.5-fold and 6-fold, respectively, for the truncated receptor relative to the wild-type MOR. Similarly, the affinities of the opioid peptide agonists, DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), beta-endorphin, and DADL (Tyr-D-Ala-Gly-Phe-D-Leu), for the DeltaN64 receptor were decreased from 3- to 8-fold as a result of the deletion. In contrast, the affinities of the alkaloid agonists, methadone and fentanyl, and the peptide agonists, endomorphin 1 and endomorphin 2, for the truncated receptor relative to MOR were reduced dramatically by 20- to 60-fold. MOR is glycosylated when expressed in HEK 293 cells; however, analysis of N-glycosidase F-treated membranes indicated that N-glycan chains within the amino-terminal domain of MOR do not contribute significantly to ligand affinities. These results indicate that amino acid residues within the amino-terminal domain of MOR play a crucial role in the composition of the binding pocket for a select group of agonists.

Changes in glutamate receptor and proenkephalin gene expression after kindled seizures

Changes in gene expression after kindled seizures were examined using microdissection of discrete brain areas and Northern and slot blot analyses. Experimental animals were kindled with either of two protocols: (1) a paradigm in which 50 Hz/10 s stimulus trains were delivered every 30 min through hippocampal electrodes (12 stimulations every other day for 4 days) and (2) a traditional approach in which 50 Hz/10 s stimulus trains were given to the hippocampus three times daily for 16 days. Rats were sacrificed 24 h or 30 days after the last kindled seizure. We first examined the possibility that kindling may affect transcription of mRNA for neurotransmitter receptors. We found significant decreases (22-58%) in AMPA/kainate activated glutamate receptor mRNAs (GluR1, -2, -3 mRNAs) in hippocampus, amygdala/entorhinal cortex and in frontoparietal cortex 24 h but not 30 days after rapidly kindled seizures. However, changes in GABA receptor alpha 1, alpha 2, alpha 4 or beta 1 mRNAs were not observed in any brain region 30 days after traditional kindling or 24 h after rapidly kindled seizures. In addition, we tested whether changes in the expression of proenkephalin could be detected after kindling. We found significant increases (1.7-10 fold) in proenkephalin mRNA in the frontoparietal cortex, hippocampus and in the amygdala/entorhinal cortex 24 h but not 30 days after rapidly kindled seizures. Our findings suggest that changes in glutamate receptor and proenkephalin gene expression are robust, acute sequelae to kindled seizures and may be involved in kindling.

Studies on Inhibition of and Opioid Receptor Binding by Dithiothreitol and N-Ethylmaleimide His IS CRITICAL FOR μ OPIOID RECEPTOR BINDING AND INACTIVATION BY N-ETHYLMALEIMIDE

The sensitivity of μ and receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. μ receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC of N-ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the μ receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-ethylmaleimide. Serine substitution of His in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N-ethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the μ opioid receptor.

Glucocorticoid-mediated down regulation of c-fos mRNA in C6 glioma cells: lack of correlation with proenkephalin mRNA

We investigate the linkage between the transcriptional factor, c-fos, and expression of proenkephalin in rat C6 glioma cells. C6 cells contained abundant levels of c-fos mRNA. Treatment of cells with dexamethasone resulted in a 10-fold decline in c-fos transcripts and a small increase in proenkephalin mRNA. Combined exposure to dexamethasone and isoproterenol also induced a decrease in c-fos mRNA while proenkephalin mRNA increased 8-fold. Treatment of the C6 cells with phorbol 12-myristate 13-acetate caused a 13-fold increase in c-fos expression 0.5 h after administration and a decrease in proenkephalin mRNA. These data indicate that c-fos and proenkephalin mRNA are not regulated in a sequential, parallel manner, that newly synthesized c-fos is not the determining factor controlling proenkephalin gene regulation, and that c-fos expression is under negative control by glucocorticoids.

Stimulation of c-fos and c-jun gene expression and down-regulation of proenkephalin gene expression in C6 glioma cells by endothelin-1.

The linkage between the transmembrane signal transduction system utilized by endothelin and alterations in gene expression has been investigated in C6 glioma cells. Treatment of C6 cells with endothelin-1 caused a rapid and transient 5-fold increase in c-fos and c-jun mRNA levels, followed by a decrease at 4 h. Dose-response studies indicated that 1 nM endothelin-1 caused half-maximal induction of c-fos mRNA 0.5 h after treatment and that maximal induction was elicited with a concentration of 10 nM. Actinomycin D totally abolished the rapid increase in c-fos mRNA caused by endothelin, indicating that the effect is at the transcriptional level. Endothelin-1 caused a decrease in proenkephalin mRNA to 50% of control levels at 4 h after treatment and had no effect on histone H4 mRNA over a 24 h period that was examined. These data indicate that receptor binding of endothelin-1 leads to rapid changes in the expression of immediate-early response genes which may cause more prolonged changes in the expression of AP-1 and/or CREB target genes in the nervous system.

Molecular cloning, sequence analysis and translation of proenkephalin mRNA from rat heart.

Proenkephalin mRNA is abundant in rat cardiac ventricles but surprisingly low levels of opioid peptides or precursor forms derived from proenkephalin are present in tissue extracts. Proenkephalin mRNA in rat heart was characterized at the molecular level with the use of cDNA sequencing, in vitro translation, and primer extension. Two positive proenkephalin cDNA clones were obtained by screening approx. 20,000 recombinant phages from a heart cDNA library. Sequence analysis of the cDNA clones indicated that the heart transcript was the same form as in rat brain, but differed from the germ cell-specific testis transcript that utilizes a different transcriptional start site. Heart proenkephalin cRNA translated efficiently, resulting in the synthesis of a 35 kDa protein that was immunoprecipitated by an antibody specific to the protein. The transcriptional initiation sites utilized in the heart were the same as in the brain, based on primer extension studies. These data suggest that the proenkephalin transcript found in abundance in rat heart is the same form as found in the brain, and differs from the testis-type transcript. We conclude that the scant level of proenkephalin-derived peptides in the heart is not due to an intrinsic inability of the proenkephalin transcript to translate.

Pharmacological profiles of selective non-peptidic δ opioid receptor ligands

Abstract Several non-peptidic opioids have been synthesized recently as part of a program to develop selective δ receptor agonists. In this study, the affinities of a set of compounds for cloned δ and μ opioid receptors expressed in HEK 293 cell lines were determined by competition analysis of [3H]bremazocine binding to membrane preparations. All compounds studied exhibited high affinity and selectivity, with apparent dissociation constants in the range of 0.6–1.7 nM for the δ opioid receptor and 240–1165 nM for the μ opioid receptor. We next sought to determine which domain of the δ receptor was critical for mediating the highly selective binding by analysis of ligand affinities for μ/δ receptor chimeras. Receptor binding profiles suggested that a critical site of receptor/ligand interaction was located between transmembrane domain 5 (TM5) and TM7 of the δ receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the μ opioid receptor, led to a spectrum of effects on affinities, depending on the ligand tested. Affinities of SB 219825 and SB 222941 were particularly sensitive to the substitution, displaying a 50-fold and 70-fold decrease in affinity, respectively. Activities of the δ receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing δ opioid receptors with selective ligands induced phosphorylation of MAP kinase, although the non-peptidic ligands were less efficacious than the enkephalin derivative DADL (Tyr- d -Ala-Gly-Phe- d -Leu). Similarly, chronic exposure of HEK 293 cells expressing δ opioid receptors with selective, non-peptidic ligands, with the exception of SB 206848, caused receptor down-regulation, however, the SB compounds were less efficacious than DADL.