In vitro and in vivo expression of opioid and sigma receptors in rat C6 glioma and mouse N18TG2 neuroblastoma cells.

Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for delta opioid binding using [3H][D-Ala2,D-Leu5]enkephalin and for sigma 1 and sigma 2 binding with 1,3-[3H]di-o-tolylguanidine in the presence and absence of 1 microM pentazocine. Receptor density (Bmax) and affinity (KD) were estimated by homologous competition binding assays. Opioid and sigma Bmax values in the solid tumors were significantly lower than their original levels in vitro. KD values for opioid/sigma ligands were similar in vitro and in vivo. With successive passages in the murine host, delta opioid and sigma 1 binding of the neuroblastoma-derived solid tumors became undetectable. In contrast, sigma 2 receptor Bmax values were unchanged with successive passages of the neuroblastoma-derived tumors and doubled in the nude mouse-borne gliomas. When neuroblastoma-derived solid tumors that were devoid of delta opioid binding were returned to culture, opioid receptors appeared to be up-regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for sigma binding was more complex, with the sigma 2 response in late passages of the glioma being reminiscent of the formerly observed increase in number of sigma sites in transformed human meninges, kidney, and colon tissue.

Differential development of beta-endorphin and mu opioid binding sites in mouse brain.

Mouse brains of various ages from embryonal day 14 (E14) to adult were analyzed for opioid receptor binding using the enkephalin analog Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and the opiate alkaloid dihydromorphine (DHM) as mu-selective radioligands. Binding parameters were estimated from homologous and heterologous competition binding curves. During the postnatal period, Kd values for [3H]DAMGE did not change but Bmax values (fmol/mg protein) increased 2.7 fold from postnatal day 3 (P3) to P7. Minor receptor density fluctuations were evident from P7 to adult. Similar results were obtained with [3H]DHM. In contrast, estimation of total mu binding sites (fmol/brain) revealed a continuous rise from P3 to the adult. The postnatal developmental profile of total mu binding sites was comparable to the weight gain of mouse brain and the increase in protein content. In contrast, during the same period beta-endorphin immunoreactivity (IR) levels undergo an increase that is inversely proportional to mu opioid receptor Bmax values. [3H]DAMGE binding to E14 membrane preparations was inhibited to a greater extent by Gpp(NH)p than that to P1 or adult. Additional characterization of mu receptors was accomplished by heterologous competition binding assays. IC50 values for beta-endorphin in competition with [3H]DHM and [3H]DAMGE were age dependent and differed for the two radioligands. These results suggest that mu receptor selectivity for mu-specific peptide and alkaloid ligands changes as a function of age.

Overexpression of sigma receptors in nonneural human tumors.

Previous data indicated that opioid receptors occur in both neural and nonneural human tumors. However, it has recently been shown that some of the putative opioid binding may be attributable to sigma sites. In this study the occurrence of sigma and opioid receptors in nonneural human tumors was assessed. The neoplasms included renal and colon carcinomas and a sarcoma. [3H]1,3-di-o-tolylguanidine was used to assay sigma receptors by homologous competition binding assays, which when analyzed provided dissociation constant and receptor density values. Opioid binding was measured with [3H]-(-)-ethylketocyclazocine, a ligand which interacts with mu, delta, and kappa subtypes. Fresh surgical specimens were obtained from 9 human neoplasms, selected for their large size, and compared with nonmalignant tissues. All 9 tumors contained sigma sites, and dissociation constant values were within the range of 27-83 nM. Occasionally, two-site fit the data better than one-site binding, suggesting the presence of multiple sigma sites. Opioid binding was not detected. Intratumoral variability was evaluated by sampling several locations on the periphery of the mass and one in the center. Each of the samples was bisected, with a portion reserved for histological examination to correlate morphological features with receptor data. Changes in sigma binding were not associated with the extent of fibrosis, viability, or necrosis. Receptor density values displayed moderate intra- and intertumoral variation (coefficients of variation, 8-39 and 27-49%, respectively). More important, sigma binding in tumors was found to be greater than or equal to 2-fold higher than that of control nonmalignant tissue.

Age-dependent changes in the subcellular distribution of rat brain mu-opioid receptors and GTP binding regulatory proteins.

The relative subcellular distributions of mu-opioid receptors and guanine nucleotide binding regulatory proteins (G proteins) in 1-day-old (P1) and adult rat forebrain were compared. Light membranes (LMs) were resolved from heavy membranes (HM) by sucrose density gradient centrifugation. Marker enzyme analyses indicated that LMs contained most of the endoplasmic reticulum and Golgi complexes, whereas HMs were enriched in plasma membranes. Binding distribution and properties of mu-opioid sites were assessed using [3H] [D-Ala2,Me-Phe4,Gly-ol5]enkephalin. P1 LMs possessed 43% of the total mu-opioid binding detected compared to 16% in the adult. Although NaCl inhibited mu binding in LMs to a greater extent than in HMs, age-dependent differences were not observed. P1 LM mu binding possessed greater sensitivity to 5'-guanylylimidodiphosphate than their adult counterpart. Moreover, P1 LMs contained more Go alpha protein than P1 HMs or adult LMs, as demonstrated by immunoblotting with antisera against Go alpha after one- or two-dimensional gel electrophoresis. These results suggest that P1 LMs contain a greater proportion of newly synthesized intracellular mu sites than adult LMs.

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.

A monoclonal anti-idiotypic antibody to mu and delta opioid receptors.

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.

The prenatal development profile of expression of opioid peptides and receptors in the mouse brain.

Although the postnatal development of opioid systems of mammalian brain has been well studied, little is known about the ontogeny of and relationship between embryonic (E) opioid peptides and their receptors. Moreover, a simultaneous assessment of levels of the 3 classes of opioid peptides and their putative receptors during embryonal development has not been made. To this end, the ontogeny of opioid peptides and receptors in mouse brain were examined during the period E11.5 to postnatal day 1 (P1). Met-enkephalin, dynorphin and beta-endorphin immunoreactivity were detected before their putative opioid receptors. beta-Endorphin can be discerned as early as E11.5, whereas mu binding was first observed at E12.5. Although dynorphin and Met-enkephalin were measurable at the same time as beta-endorphin, kappa-receptors were not detected until E14.5 and delta sites were not found at all prenatally. Differences in immunoreactivity levels of the 3 peptides occur with dynorphin being lower than Met-enkephalin and beta-endorphin, consistent with a low Bmax for kappa binding. Expression of the 3 opioid peptides as well as mu and kappa opioid receptors rapidly increase in parallel from E14.5 to E18.5. Interestingly, levels of beta-endorphin diminish by P1, the stage at which a sharp rise of mu receptors occurs. In a comparative study of the binding of beta-endorphin 1-31, its truncated form (1-27) and their N-acetyl derivatives to E14.5 brain membranes, beta-endorphin 1-31 exhibited the highest affinity.

Sigma and opioid receptors in human brain tumors.

Human brain tumors (obtained as surgical specimens) and nude mouse-borne human neuroblastomas and gliomas were analyzed for sigma and opioid receptor content. Sigma binding was assessed using [3H]1,3-di-o-tolylguanidine (DTG), whereas opoid receptor subtypes were measured with tritiated forms of the following: mu, [D-ala2,mePhe4,gly-ol5]enkephalin (DAMGE); kappa, ethylketocyclazocine (EKC) or U69,593; delta, [D-pen2,D-pen5]enkephalin (DPDPE) or [D-ala2,D-leu5]enkephalin (DADLE) with mu suppressor present. Binding parameters were estimated by homologous displacement assays followed by analysis using the LIGAND program. Sigma binding was detected in 15 of 16 tumors examined with very high levels (pmol/mg protein) found in a brain metastasis from an adenocarcinoma of lung and a human neuroblastoma (SK-N-MC) passaged in nude mice. kappa opioid receptor binding was detected in 4 of 4 glioblastoma multiforme specimens and 2 of 2 human astrocytoma cell lines tested but not in the other brain tumors analyzed.

Unscheduled DNA synthesis related to transformation grade of human urothelial cells.

Unscheduled DNA synthesis (UDS) was studied by quantitative autoradiography in human urothelial cells of three transformation grades (TGr I-III). Cells incubated in arginine-free medium supplemented with hydroxyurea showed dose-dependent UDS after administration of agents injurious to DNA, while the scheduled synthesis of DNA was nearly totally suppressed. UDS was demonstrated after treatment with the ultimate carcinogen N-methyl-N-nitroso-N'-nitroguanidine (CAS: 70-25-7) or with the procarcinogen 4-nitroquinoline-1-oxide (4-NQO; CAS: 56-57-5). In cultures treated with benzo[a]pyrene (CAS: 50-32-8), which requires a different activation system than that for 4-NQO, UDS was less pronounced. In 9 cell lines the average rates of UDS were inversely related to TGr. Two cell lines showed a different pattern.