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Biomedical subjects

Gavril W Pasternak

Publications and source records attributed to Gavril W Pasternak.

At least 19 recordsLinked to original sources
PubMed2006

Codeine and 6-acetylcodeine analgesia in mice.

1. Acetylation of morphine at the 6-position changes its pharmacology. To see if similar changes are seen with codeine, we examined the analgesic actions of codeine and 6-acetylcodeine. 2. Like codeine, 6-acetylcodeine is an effective analgesic systemically, supraspinally and spinally, with a potency approximately a third that of codeine. 3. The sensitivity of 6-acetylcodeine analgesia to the mu-selective antagonists beta-FNA and naloxonazine confirmed its classification as a mu opioid. However, it differed from the other mu analgesics in other paradigms. 4. Antisense mapping revealed the sensitivity of 6-acetylcodeine to probes targeting exons 1 and 2 of the mu opioid receptor gene (Oprm), a profile distinct from either codeine or morphine. Although heroin analgesia also is sensitive to antisense targeting exons 1 and 2, heroin analgesia also is sensitive to the antagonist 3-O-methylnaltrexone, while 6-acetylcodeine analgesia is not. 5. Thus, 6-acetylcodeine is an effective mu opioid analgesic with a distinct pharmacological profile.

Analgesia↗
PubMed2006

Separation of binding affinity and intrinsic activity of the potent mu-opioid 14-methoxymetopon.

Receptor binding studies of 5,14-O-dimethyloxymorphone (14-methoxymetopon) in brain membranes have established its high affinity for mu-binding sites, but its analgesic potency far exceeds the modest increase in binding affinity relative to other opioids. The current study has established the selectivity of [(3)H]14-methoxymetopon for mu sites in calf striatal membranes and for a number of full-length splice variants of the cloned murine mu-opioid receptor 1 (mMOR-1) in transfected cell lines. The binding affinity of [(3)H]14-methoxymetopon for the variants expressed in Chinese hamster ovary cells was quite high, with K(D) values around 0.2 nM for all of the variants with the exception of mMOR-1F (K(D) of 1.2 nM). The affinity for most of the expressed variants was greater than that seen in the brain membranes (K(D) of 0.99 nM). Functionally, in guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays with the MOR-1 variants, 14-methoxymetopon and the mu-opioid peptide [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) showed similar efficacies, as determined by maximal stimulation, but 14-methoxymetopon was up to 65-fold more potent than DAMGO. The greatest difference was seen with mMOR-1E and the least with mMOR-1C, which displayed only a 10-fold difference. These potency differences in the stimulation of [(35)S]GTPgammaS binding far exceeded the differences in binding affinity. The differences between 14-methoxymetopon and DAMGO remained after normalizing the potency shifts based upon receptor binding affinities and varied from 1.2-fold with mMOR-1C to 21-fold for mMOR-1 and 42-fold with mMOR-1F. Thus, 14-methoxymetopon is a potent agonist against all of the mMOR-1 splice variants, but its potency ranged widely despite similar binding affinities for most of the variants and may give insight into its unusual pharmacological profile.

Animals↗
PubMed2006

Immunohistochemical labeling of the mu opioid receptor carboxy terminal splice variant mMOR-1B4 in the mouse central nervous system.

The mu opioid receptor gene Oprm is alternatively spliced into many variants, providing for the multiplicity of mu opioid receptor subtypes. One of the mouse variants, mMOR-1B4, is unique in that it displays high affinity towards a wide range of mu opioid receptor antagonists, but poor affinity towards most classical mu opioid agonists. The present study examined the immunohistochemical distribution of the mMOR-1B4 variant in mouse brain and spinal cord. mMOR-1B4-like immunoreactivity (mMOR-1B4-LI) was enriched in many regions of the brain, spinal cord and in the dorsal root ganglia. Some of the structures showing prominent mMOR-1B4-LI include the olfactory bulb, cerebral cortex, bed nucleus of stria terminalis, hippocampus, habenular nucleus, amygdala, thalamus, hypothalamus, medium eminence, substantia nigra, ventral tegmental area, oculomotor nucleus, red nucleus, raphe nuclei, periaqueductal gray, locus coeruleus, trigeminal nucleus, reticular formation, area postrema and Purkinje cell layer and deep nuclei of cerebellum. mMOR-1B4-LI was present in afferent neurons of the dorsal root ganglia and their projecting fibers into the superficial laminae of the spinal dorsal horn. Some motor neurons in the anterior horn of the spinal cord also were immunopositive. The overall distribution of mMOR-1B4-LI in the central nervous system is distinguishable from previously characterized variants such as MOR-1-LI, MOR-1C-LI and exon-11-LI. These studies provide evidence for the region- and neuron-specific processing of the Oprm gene and support the possibility of functional differences among the variants.

Alternative Splicing↗
PubMed2005

The peripheral-type benzodiazepine receptor and tumorigenicity: isoquinoline binding protein (IBP) antisense knockdown in the C6 glioma cell line.

Peripheral-type benzodiazepine receptors (PBR) are constituted by three protein components, the isoquinoline binding protein (IBP), the voltage-dependent anion channel (VDAC), and the adenine nucleotide transporter (ANT). Recently, we found that high levels of PBR ligand binding in glioma cell lines correlate with in vitro tumorigenicity. To study whether enhanced PBR expression is causative or in response to cancer, we genetically modified C6 glioma cells. Antisense knockdown of the IBP resulted in more than 50% reductions in PBR ligand binding both in the mitochondrial and whole cell fractions, accompanied by similar reductions in IBP levels in these respective fractions. The IBP knockdown was accompanied by a 25% increase in cell number in confluent cultures. This correlated with an 8-fold increase in in vitro tumorigenicity, as assessed by anchorage independent growth. Cell cycle analysis indicated that knockdown of the IBP resulted in a 60% reduction in the number of cells in the pre-G1 apoptosis phase. This paralleled the reduction seen in apoptosis and cell death shown by DNA fragmentation and Trypan blue assays, respectively. Furthermore, knockdown of the IBP appeared to prevent induction of apoptosis by the antineoplastic agent, erucylphosphocholine. In addition, IBP knockdown prevented processing of the caspase 3 component of the apoptosis cascade by the erucylphosphocholine congener, erucylphospho-N,N,N-trimethylammonium. In conclusion, our results suggest that enhanced IBP expression, including enhanced PBR ligand binding, such as occurring in untreated C6 glioma cells, may provide a mechanism to increase apoptotic rates of cancer cells.

Animals↗
PubMed2005

Identification of four novel exon 5 splice variants of the mouse mu-opioid receptor gene: functional consequences of C-terminal splicing.

The rat mu-opioid receptor clone in which novel exon 5 was found in the place of exon 4 (MOR-1B) was one of the first MOR-1 variants described. We now have identified the mouse homolog of the rat MOR-1B as well as four additional variants derived from splicing from exon 3 into different sites within exon 5. The sequences of all of the variants were identical except for the intracellular tip of the C terminus encoded by exon 5, where each variant predicted a unique amino acid sequence ranging from 2 to 39 amino acids. All of the mMOR-1B variants were selective for mu-opioids in receptor-binding assays, as anticipated, because they all have identical binding pockets defined by the transmembrane domains. However, the relative potency and efficacy of mu-agonists to each other varied from variant to variant in guanosine 5'-O-(3-[35S]thio)triphosphate-binding studies, as shown by morphine-6beta-glucuronide, which was the most efficacious agent against mouse MOR-1B1 (mMOR-1B1) and the least efficacious agent against mMOR-1B2. mMOR-1B4 was quite unusual. Although mMOR-1B4 was mu-selective in receptor-binding studies and antagonists labeled mMOR-1B4 well, the binding affinities of most of the mu-agonists were far lower than those seen with mMOR-1, suggesting that the 39 amino acids at the C terminus of mMOR-1B4 influences the conformation of the receptor and its ligand recognition site itself either directly or through its interactions with other proteins. In conclusion, alterations in the amino acid sequence of the C terminus do not alter the mu-specificity of the receptor but they can influence the binding characteristics, efficacy, and potency of mu-opioids.

Animals↗
PubMed2005

Selective potentiation of opioid analgesia by nonsteroidal anti-inflammatory drugs.

Opioids are often used in conjunction with nonsteroidal anti-inflammatory drugs (NSAIDs) in the treatment of moderate to severe pain. In this study we have examined interactions between these two classes of drugs. NSAIDs are inactive in the radiant heat tail-flick test, an assay of moderate to severe pain in which opioids are effective. In this assay, ibuprofen potentiated the analgesic actions of hydrocodone and oxycodone, shifting their ED(50) values by 2.5-fold and 4.6-fold despite its inactivity when given alone. These opioid/NSAID interactions were dependent upon both the opioid and the NSAID. Neither aspirin nor ketorolac influenced hydrocodone actions in this model and ibuprofen did not potentiate fentanyl or morphine analgesia. Together, these studies demonstrate potent interactions between selected combinations of opioids and NSAIDS and may help explain the clinical utility of combinations. However, the findings also illustrate differences between the drugs within each class.

Analgesics, Opioid↗
PubMed2005

Affinity labeling mu opioid receptors with novel radioligands.

1. A series of novel opiate ligands based upon 6alpha-naloxamine have been examined in opioid receptor binding assays. 2. Coupling an ethylamine spacer alone to 6-alpha-naloxamine gave a compound with relatively poor affinity for mu opioid receptors compared to naloxone, although it retained high affinity for kappa1 opioid receptors. Coupling a benzoyl group significantly increased the affinity. The presence at the 4-position of the benzoyl moiety of an amino-(NalAmiBen) or an azido-substituent (NalAziBen) did not significantly effect the affinity at mu receptors. However, iodinating the benzoyl moiety at the 3-position increased the affinity of the derivatives. 3. Two compounds were radiolabeled and evaluated in receptor binding assays. Both radioligands labeled sites in CHO cells stably transfected with the mouse MOR-1 clone. The amino coupound [125I]NalAmiBen and the azido derivative [125I]NalAziBen reversibly bound to membranes from CHO cells transfected with MOR-1 with high affinity in the dark. Exposure of [125I]NalAmiBen to UV did not alter the reversibility of binding, but exposure of [125I]NalAziBen to UV light led to the covalent coupling of the radioligand to the receptor. When run on SDS-PAGE, [125I]NalAziBen binding showed a band at approximately 70-80 kDa. A control corresponding to nonspecific binding failed to reveal any labeling. No bands were observed from membranes labeled with [125I]NalAmiBen.

Affinity Labels↗
PubMed2005

Molecular biology of opioid analgesia.

Opioids provide excellent pain relief in most patients. Yet the responses of patients to individual opioids can vary markedly, even among the mu opioids. Understanding this variability would greatly enhance our ability to treat patients appropriately. Classical pharmacological studies have long implied the existence of multiple subtypes of mu opioid receptors. More recently, a number of variants of the cloned mu opioid receptor have been described. These variants all show the same selectivity for mu opioids, confirming their classification as mu opioid receptors. Yet, they differ in their functional activation by opioids as well as in their localization within cells and regions in the brain. These multiple mu opioid receptors may help explain the range of responses seen clinically among patients for the various opioid drugs.

Analgesics, Opioid↗
PubMed2004

Pharmacological characterization of dihydromorphine, 6-acetyldihydromorphine and dihydroheroin analgesia and their differentiation from morphine.

The present study examined the pharmacology of dihydromorphine, 6-acetyldihydromorphine and dihydroheroin (3,6-diacetyldihydromorphine). Like morphine, dihydromorphine and its acetylated derivatives all were highly selective mu-opioids in receptor binding assays. All the compounds were potent mu-selective analgesics, as shown by their sensitivity towards the mu-selective opioid receptor antagonists naloxonazine and beta-funaltrexamine. However, the actions of dihydromorphine and its analogs were readily distinguished from those of morphine, differences that were surprising in view of the very limited structural differences among them that consisted of only the reduction of the 7,8-double bond. Like heroin and morphine-6beta-glucuronide, the analgesic actions of dihydromorphine and its two acetylated derivatives were antagonized by 3-O-methylnaltrexone at a dose that was inactive against morphine analgesia. Antisense mapping also distinguished between morphine and the dihydromorphine compounds. Antisense oligodeoxynucleotides targeting exon 2 of the cloned MOR-1 gene decreased dihydromorphine analgesia and that of its acetylated derivatives, but not morphine analgesia. Conversely, the exon 1 antisense that effectively lowered morphine analgesia was inactive against dihydromorphine and its analogs. Finally, dihydromorphine and its analogs retained their analgesic activity in a mouse model of morphine tolerance, consistent with incomplete cross-tolerance. Together, these findings imply that the mu-opioid receptor mechanisms mediating the analgesic actions of dihydromorphine and its acetylated analogs are distinct from morphine and more similar to those of heroin and morphine-6beta-glucuronide.

Analgesics, Opioid↗
PubMed2004

Functional analysis of MOR-1 splice variants of the mouse mu opioid receptor gene Oprm.

A series of mu opioid receptor gene Oprm splice variants have been reported that differ only at their C-terminus. These variants all contain exons 1, 2, and 3 of the gene, the exons responsible for coding all seven transmembrane domains. Whereas MOR-1 also has exon 4 that encodes for an additional 12 amino acids at the tip of the C-terminus, the other MOR-1 variants have unique amino acid sequences distinct from those in MOR-1 due to alternative splicing. All these variants are mu-selective in binding assays. The current study explored the ability of these variants to stimulate [35S]GTPgammaS binding to assess them functionally. Only mu opioids stimulated [35S]GTPgammaS binding. Among the mu opioids we noted marked differences in their maximal stimulation among the clones. This was most prominent with beta-endorphin, which stimulated [35S]GTPgammaS binding in the MOR-1E expressing cells to a greater degree than [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO; 130%) and was far less effective than DAMGO in MOR-1C cells (44%). The rank order of maximal stimulation of the drugs varied among the clones as well. Dynorphin A, beta-endorphin and morphine were most effective in stimulating [35S]GTPgammaS binding in MOR-1E, while M6G and fentanyl were most effective in MOR-1 expressing cells. The potency (EC50) of some of the drugs also varied extensively among the clones, with a poor correlation between the potency of the drugs to stimulate [35S]GTPgammaS binding and their binding affinity. Together, these findings reveal marked functional differences among the variants that only can be explained by their structural differences at the tip of their C-terminus.

Alternative Splicing↗
PubMed2004

Multiple opiate receptors: déjà vu all over again.

The concept of multiple opioid receptors has changed dramatically since their initial proposal by Martin nearly 40 years ago. Three major opioid receptor families have now been proposed: mu, kappa and delta. Most of the opioid analgesics used clinically selectively bind to mu opioid receptors. Yet, clinicians have long appreciated subtle, but significant, differences in their pharmacology. These observations suggested more than one mu opioid receptor mechanism of action and led us to propose multiple mu opioid receptors over 20 years ago based upon a range of pharmacological and receptor binding approaches. A mu opioid receptor, MOR-1, was cloned about a decade ago. More recent studies have now identified a number of splice variants of this clone. These splice variants may help explain the pharmacology of the mu opioids and open interesting directions for future opioid research.

Animals↗
PubMed2004

Identification of three new alternatively spliced variants of the rat mu opioid receptor gene: dissociation of affinity and efficacy.

Mu opioid receptors mediate the pharmacological actions of morphine and morphine-like drugs, such as heroin. The mouse and human Oprm genes undergo splicing. In these present studies, we have identified and characterized three new MOR-1 splice variants from the rat Oprm gene. Using an RT-PCR approach, we isolated the new exons 7, 8 and 9 downstream of exon 3. The rat exons 7 and 9 were homologous to the mouse exons 7 and 9 while the rat exon 8 was not. Northern blot analysis with the new exon probes showed distinctive and abundant transcripts of the variants in the rat brain. Full-length cDNA clones containing the new exons, rMOR-1C1, rMOR-1C2 and rMOR-1D were obtained using an RT-PCR approach. Each contained the same exons 1, 2 and 3 as the original rMOR-1, followed by different combinations of the new exons in place of exon 4. In addition, we also isolated another new variant, rMOR-1A, which contains only exons 1, 2 and 3, and is homologous to the human variant MOR-1A previously identified. All the variants were highly mu-selective in binding studies with little difference in affinities for the mu ligands among the variants. However, functional evaluation of assessments of the variants using agonist stimulated [(35)S]GTPgammaS binding assays revealed marked differences among the variants, both in terms of potency and efficacy of the drugs. The relative efficacy of a series of mu opioids to each other varied depending upon the variant studied. Efficacy in the [(35)S]GTPgammaS assay did not correlate with either receptor binding affinity or with potency. Thus, selectivity of opioid action might be achieved by designing compounds with varying efficacies at different MOR-1 variants.

Alternative Splicing↗
PubMed2003

Differential sensitivities of mouse strains to morphine and [Dmt1]DALDA analgesia.

[Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2)), is a highly potent and selective mu-opioid agonist. Nevertheless, systemic [Dmt(1)]DALDA retained its analgesic actions in MOR-1 knockout animals and CXBK mice despite the inactivity of morphine in these mice. [Dmt(1)]DALDA was 6-fold less potent in C57BL/6J mice than in CD-1 mice, whereas morphine potency did not differ between the two strains. Thus, [Dmt(1)]DALDA is a highly selective mu-opioid analgesic with significant pharmacological differences with the prototypic mu-opioid morphine.

Analgesics, Opioid↗
PubMed2003

Improved brain uptake and pharmacological activity of dalargin using a peptide-vector-mediated strategy.

The blood-brain barrier restricts the passage of substances into the brain. Neuropeptides, such as enkephalins, cannot be delivered into the brain when given systemically because of this barrier. Therefore, there is a need to develop efficient transport systems to deliver these drugs to the brain. Recently, we have demonstrated that conjugation of doxorubicin or penicillin to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated the enkephalin analog dalargin with two different peptide vectors, SynB1 and SynB3, to improve its brain delivery and its pharmacological effect. We show by in situ brain perfusion that vectorization markedly enhances the brain uptake of dalargin. We also show using the hot-plate model that this enhancement in brain uptake results in a significant improvement in the observed antinociceptive effect of dalargin. These results support the usefulness of peptide-mediated strategies for improving the availability and efficacy of central nervous system drugs.

Analgesia↗
PubMed2003

Characterization of the binding of [3H][Dmt1]H-Dmt-D-Arg-Phe-Lys-NH2, a highly potent opioid peptide.

The dermorphin-derived peptide [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt, 2',6'-dimethyltyrosine) labels mu-opioid receptors with high affinity and selectivity in receptor binding assays. In previous studies, [Dmt1]DALDA displayed a mechanism of action distinct from that of morphine, as evidenced by its insensitivity to antisense probes reducing morphine analgesia and incomplete cross tolerance to morphine. In an effort to further elucidate the unusual mechanism of action, [3H][Dmt1]DALDA has been synthesized and its binding profile studied. [3H][Dmt1]DALDA binding was high affinity (KD = 0.22 nM) and showed a regional distribution consistent with mu-receptors with highest levels in calf striatal membranes. [3H][Dmt1]DALDA binding was far less sensitive than [3H][d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) to the effects of divalent and sodium cations and guanine nucleotides, although NaCl and guanosine 5'-(beta,gamma-imido)triphosphate together reduced specific [3H][Dmt1]DALDA binding levels by almost 75%. Competition studies confirmed the mu-selectivity of the binding, with Ki values that were not appreciably different from those seen against [3H]DAMGO. In guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding assays in brain and spinal cord membranes, [Dmt1]DALDA was more potent than DAMGO, but showed plateaus suggestive of a partial agonist. [Dmt1]DALDA bound to mu-opioid receptor clone 1 (MOR-1) and its splice variants with high affinity. Unlike [3H]DAMGO, [3H][Dmt1]DALDA seemed to label both agonist and antagonist conformations of MOR-1 expressed in Chinese hamster ovary cells. In [35S]GTPgammaS assays [Dmt1]DALDA showed high efficacy with all the MOR-1 variants, but its potency (EC50) varied markedly among some of the splice variants despite similar affinities in receptor binding assays. Although [3H][Dmt1]DALDA is a very potent mu-selective analgesic, its binding characteristics and its ability to stimulate GTPgammaS binding differed from that of the classical mu-opioid peptide DAMGO.

Alternative Splicing↗
PubMed2003

2,6-Dimethyltyrosine analogues of a stereodiversified ligand library: highly potent, selective, non-peptidic mu opioid receptor agonists.

We recently reported the use of an exhaustively stereodiversified library based on endomorphin-2 (1) to discover mu opioid receptor (MOR) ligands of type 2-4. Here, we report the synthesis and evaluation of 2,6-dimethyltyrosine analogues 5-10. These analogues showed improved affinity for MOR relative to 2-4. In the cases of 5 and 6, we synthesized and evaluated five stereoisomers of each, thereby discovering stereoisomers with unexpected potency, selectivity, and efficacy. These results illustrate the utility of acyclic, stereodiverse libraries.

Animals↗
PubMed2003

Identification and characterization of two new human mu opioid receptor splice variants, hMOR-1O and hMOR-1X.

The mouse gene encoding the mu opioid receptor, Oprm, undergoes extensive alternatively splicing, with 14 variants having been identified. However, only one variant of human mu opioid receptor gene (Oprm), MOR-1A, has been described. We now report two novel splice variants of the human Oprm gene, hMOR-1O and hMOR-1X. The full-length cDNAs of hMOR-1O and hMO-1X contained the same exons 1, 2, and 3 as the original hMOR-1, but with exon O or exon X as the alternative fourth exon, respectively. Northern blots revealed several bands with the exon O probe in both human neuroblastoma BE(2)C cells and human brain and a single band (5.5kb) with the exon X probe in selected human brain regions. When transfected into CHO cells, both variants showed high selectivity for mu opioids in binding assays. These two new human mu opioid receptors are the first human MOR-1 variants containing new exons and suggest that the complex splicing present in mice may extend to humans.

Alternative Splicing↗