Biomedical subjects

J Ryan-Moro

Publications and source records attributed to J Ryan-Moro.

6 recordsLinked to original sources

PubMed1999

[(125)I]orphanin FQ/nociceptin binding in Raji cells.

Western blots using an antibody which recognizes the orphanin FQ/nociceptin (OFQ/N) receptor reveals a band at approximately 69 kD in several cell lines, including the Raji human B cell lymphoma cell line. RT-PCR confirms the presence of this receptor in the Raji cells. Binding studies revealed a high affinity [(125)I][Tyr(14)]OFQ/N site in the Raji cells. The affinity of [(125)I][Tyr(14)]OFQ/N in the Raji cells (K(D) 68.4 pM) was similar to that in the transfected receptor (K(D) 36.7 pM). Its selectivity profile also was quite similar. OFQ/N competed binding quite potently (K(i) 65 pM), as did [Tyr(14)]OFQ/N (K(i) 33 pM). Traditional opioids displayed no appreciable affinity for the binding at any concentration examined, with the exception of naloxone benzoylhydrazone, which had only a very modest affinity. The receptors in the Raji cells were functionally active. OFQ/N inhibited forskolin-stimulated cyclase by 72% with an IC(50) value of approximately 1 nM.

Antibodies, Monoclonal↗

PubMed1998

Pharmacological characterization of endomorphin-1 and endomorphin-2 in mouse brain.

The recently isolated peptides endomorphin-1 and endomorphin-2 have been suggested to be the endogenous ligands for the mu receptor. In traditional opioid receptor binding assays in mouse brain homogenates, both endomorphin-1 and endomorphin-2 competed both mu1 and mu2 receptor sites quite potently. Neither compound had appreciable affinity for either delta or kappa1 receptors, confirming an earlier report. However, the two endomorphins displayed reasonable affinities for kappa3 binding sites, with Ki values between 20 and 30 nM. Both endomorphins competed 3H-[D-Ala2, MePhe4,Gly(ol)5] enkephalin binding to MOR-1 receptors expressed in CHO cells with high affinity. In mouse brain homogenates 125I-endomorphin-1 and 125I-endomorphin-2 binding was selectively competed by mu ligands. 125I-Endomorphin-1 and 125I-endomorphin-2 also labeled MOR-1 receptors expressed in CHO cells with high affinity. Autoradiography of the two 125I-labeled endomorphins demonstrated regional patterns in the brain similar to those previously observed for mu drugs. Pharmacologically, the endomorphins were potent analgesics. Although they were equipotent supraspinally, endomorphin-1 was more potent spinally. Endomorphin analgesia was effectively blocked by naloxone, as well as the mu-selective antagonists beta-funaltrexamine and naloxonazine. In CXBK mice, which are insensitive to supraspinal morphine, neither endomorphin was active, consistent with a mu mechanism of action. Finally, the endomorphins inhibited gastrointestinal transit. In conclusion, these results support the mu selectivity of these agents.

Analgesics, Opioid↗

PubMed1997

Biochemical evidence for orphanin FQ/nociceptin receptor heterogeneity in mouse brain.

A recently identified novel peptide, orphanin FQ/nociceptin (OFQ/N), is an endogenous ligand for a unique member of the cloned opioid receptor family. Saturation studies in mouse brain membranes reveal curvilinear Scatchard plots with both a higher (KD 3.8 pM, Bmax 31.6 fmol/mg protein) and a lower (KD 896 pM, Bmax 233 fmol/mg protein) affinity site in brain tissue, compared to only a single site in transfected CHO cells (KD 36 pM). Competition studies confirm the high affinity of OFQ/N for this site, but shallow Hill slopes suggest heterogeneity. Traditional opioids have poor affinity for this receptor and OFQ/N and its fragments do not label traditional opioid receptors. In brain homogenates, both OFQ/N and OFQ/N(1-11) inhibit forskolin-stimulated camp accumulation with IC50 values of 1 nM or less, an action which is readily reversed by opioid antagonists. OFQ/N(1-7) shows little activity. Together, these studies suggest the presence of heterogeneous, functionally active OFQ/N receptors in mouse brain.

Animals↗

PubMed1996

Dissociation of affinity and efficacy in KOR-3 chimeras.

KOR-3 chimeras were constructed in which the first coding exon of KOR-3 was exchanged for the corresponding first coding exon of either MOR-1 (MOR-1/KOR-3) or DOR-1 (DOR-1/KOR-3). All three clones were expressed in CHO cells and characterized with regards to their binding profiles for orphanin FQ/nociceptin (OFQ/N) and a variety of opioids as well as their functional activities in cyclase studies. 125I[Tyr14]OFQ/N labels both KOR-3 (KD 37 pM) and the MOR-1/KOR-3 chimera (KD 39 pM) equally well. Although its affinity for the DOR-1/KOR-3 chimera is quite good (KD 135 pM), it is slightly lower than the other two. Competition studies confirm the high affinity of OFQ/N for all three clones. However, several competitors clearly distinguish the chimeras from KOR-3. OFQ/N(1-11) competes KOR-3 (Ki 55 nM) over 6-fold more potently than either of the chimeras. (Ki values > 350 nM). Conversely, the modest affinity of naloxone benzoylhydrazone for KOR-3 (310 nM) is greatly increased in both the MOR-1/KOR-3 (Ki 69 nM) and DOR-1/KOR-3 (Ki 74 nM) chimeras. The remainder of the opioids tested have no appreciable affinity against any of the clones. Functionally, OFQ/N inhibits forskolin-stimulated cAMP accumulation in both the KOR-3 and the MOR-1/KOR-3 chimera by almost 40%, with IC50 values in the low nanomolar range. Little activity is seen against the DOR-1/KOR-3 chimera. Naloxone benzoylhydrazone inhibits cAMP accumulation in the KOR-3 and the DOR-1/KOR-3 chimera. Although naloxone benzoylhydrazone has higher affinity for the MOR-1/KOR-3 chimera in binding studies than KOR-3 itself, it is inactive in cyclase studies using the MOR-1/KOR-3 chimera, implying that the replacement of the first coding exon increases affinity while decreasing intrinsic activity.

Animals↗

PubMed1996

Sigma binding in a human neuroblastoma cell line.

Behaviorally, sigma1 agents modulate opioid analgesia. To examine possible mechanisms responsible for these interactions, we have identified a cell line containing both sigma1 and opioid receptors. [3H](+)-pentazocine binding in BE(2)-C human neuroblastoma cells is high affinity (KD 3.4 +/- 0.7 nM) and high density (Bmax 2.98 +/- 0.14 pmol/mg protein). Competition studies reveal a selectivity profile similar to that of sigma1 sites in guinea pig brain. (+)-Pentazocine has no effect upon either basal or forskolin-stimulated cyclase in the BE(2)-C cells, but cAMP accumulation is inhibited by the morphine, DPDPE and naloxone benzoylhydrazone. (+)-Pentazocine at concentrations as high as 10 microM does not affect this opioid effect, implying that sigma1/opioid interactions are not mediated at the level of the cell. This suggest that their behavioral interactions result from interacting neural circuits. Although (+)-pentazocine is without effect in the cyclase system, it does block carbachol-stimulated phosphoinositol turnover (IC50 6.5 +/- 1.14 microM). The specificity of the effect is confirmed by the ability of haloperidol (1 microM) to shift the IC50 value of (+)-pentazocine 2-fold to the right.

Adenylyl Cyclases↗

PubMed1995

Cloning and functional characterization through antisense mapping of a kappa 3-related opioid receptor.

We have identified a putative opioid receptor from mouse brain (KOR-3), belonging to the G protein-coupled receptor family, that is distinct from the previously cloned mu, delta, and kappa 1 receptors. Assignment of the clone to the opioid receptor family derives from both structural and functional studies. Its predicted amino acid sequence is highly homologous to that of the other opioid receptors, particularly in many of the transmembrane regions, where long stretches are identical to mu, delta, and kappa 1 receptors. Both cyclazocine and nalorphine inhibit cAMP accumulation in COS-7 cells stably expressing the clone. Northern analysis shows that the mRNA is present in brain but not in a number of other organs. Southern analysis suggests a single gene encoding the receptor. A highly selective monoclonal antibody directed against the native kappa 3 receptor recognizes, in Western analysis, the clone expressed in COS-7 cells. The in vitro translation product is also labeled by the antibody. Additional clones reveal the presence of several introns, including one in the second extracellular loop and another in the first transmembrane region. Antisense studies with an oligodeoxynucleotide directed against a region of the second extracellular loop reveal a selective blockade of kappa 3 analgesia in vivo that is not observed with a mismatch oligodeoxynucleotide based upon the antisense sequence. The mu, delta, and kappa 1 analgesia is unaffected by this antisense treatment. Antisense mapping of the clone downstream from the splice site in the first transmembrane region reveals that six different antisense oligodeoxynucleotides all block kappa 3 analgesia. In contrast, only one of an additional six different antisense oligodeoxynucleotides directed at regions upstream from this splice site is effective. This strong demarcation between the two regions raises the possibility of splice variants of the receptor. An additional clone reveals an insert in the 3' untranslated region. In conclusion, the antibody and antisense studies strongly associate KOR-3 with the kappa 3-opioid receptor, although it is not clear whether it is the kappa 3 receptor itself or a splice variant.

Amino Acid Sequence↗