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The ubiquitous intracellular Ca(2+) sensor calmodulin (CaM) regulates numerous proteins involved in cellular signaling of G protein-coupled receptors, but most known interactions between GPCRs and CaM occur downstream of the receptor. Using a sequence-based motif search, we have identified the third intracellular loop of the opioid receptor family as a possible direct contact point for interaction with CaM, in addition to its established role in G protein activation. Peptides derived from the third intracellular loop of the mu-opioid (OP(3)) receptor strongly bound CaM and were able to reduce binding interactions observed between CaM and immunopurified OP(3) receptor. Functionally, CaM reduced basal and agonist-stimulated (35)S-labeled guanosine 5'-3-O-(thio)triphosphate incorporation, a measure of G protein activation, in membranes containing recombinant OP(3) receptor. Changes in CaM membrane levels as a result of overexpression or antisense CaM suppression inversely affected basal and agonist-induced G protein activation. The ability of CaM to abolish high affinity binding sites of an agonist at OP(3) further supports the hypothesis of a direct interaction between CaM and opioid receptors. An OP(3) receptor mutant with a Lys(273) --> Ala substitution (K273A-OP(3)), an amino acid predicted to play a critical role in CaM binding based on motif structure, was found to be unaffected by changes in CaM levels but coupled more efficiently to G proteins than the wild-type receptor. Stimulation of both the OP(1) (delta-opioid) and OP(3) wild-type receptors, but not the K273A-OP(3) mutant, induced release of CaM from the plasma membrane. These results suggest that CaM directly competes with G proteins for binding to opioid receptors and that CaM may itself serve as an independent second messenger molecule that is released upon receptor stimulation.

A stable isotope dilution method is presented by which uracil (Ura) and thymine (Thy) can be determined with high precision and sensitivity at the picomole level utilizing stable isotope dilution and gas chromatography electron impact mass spectrometry in the selected ion monitoring mode. [15N2]Ura and [2H3]Thy served as internal standards. The molecular ions as well as the [M - CH3]+ion fragments of silylated Ura and Thy (Ura-TMS and Thy-TMS) were suitable for the assay which provides evidence of specificity, if identical results are obtained at both ions. Nucleosides and nucleotides of Ura and Thy were determined following quantitative hydrolysis in 6 N HCI at 180°C for two hours. Other hydrolsis procedures did not give satisfactory results. Levels of free Ura and Thy were measured in human and rat plasma after solvent extraction with a sensitivity of 20–40 pm ml-1demonstrating ready applicability of the assay method to biological samples. The potential physiological role of circulating Ura and Thy is discussed.

Previous studies measuring opioid inhibition of cyclic adenosine monophosphate in SH-SY5Y cells supported the hypothesis that continuous agonist stimulation causes a gradual conversion of the mu opioid receptor to a sensitized or constitutively active state termed mu*. Conversion to mu* was prevented by the kinase inhibitor H7, but not its close analog H8. Naloxone was proposed to act as a negative antagonist (inverse agonist) blocking mu* activity, whereas D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) appeared to act as a neutral antagonist having no effect on mu* activity. Initial in vivo results indicated that mu* activity may play a role in narcotic tolerance and dependence (Wang et al., Life Sci. 54: PL339-PL350 1994). Our study explores the pharmacology of H7 and H8, naloxone and CTAP in mice after induction of acute tolerance and dependence induced by a single s.c. dose of morphine (100 mg/kg). Physical dependence was defined by withdrawal jumping induced by i.p. naloxone injections 4 hr after the morphine dose, the time of maximal physical dependence. Neither H7 nor H8 (50 nmol or less) induced jumping, affected morphine antinociception or produced significant behavioral effects, when injected by the intracerebroventricular (i.c.v.) or intrathecal (i.th.) routes. When given 30 min before the naloxone challenge, H7, but not H8, significantly reduced naloxone jumping by i.c.v. injection. Administration of naloxone into the central nervous system, rather than by i.p. administration, required coinjection by both i.c.v. and i.th. routes to elicit full withdrawal jumping (30 nmol at each site). In contrast, the putative neutral antagonist CTAP caused little withdrawal jumping when coinjected i.c.v. and i.th., as expected if modulation of mu* activity played a role in dependence. However, CTAP was capable of partially reversing naloxone (i.p.) induced jumping when given either i.c.v. or i.th., indicating that CTAP competes with naloxone at mu*. Moreover, these results demonstrate that both spinal and supraspinal sites are required for full opioid withdrawal jumping in mice. Antinociceptive tolerance was also evaluated by determining the response to morphine in the 55 degrees C warm-water tail-flick test. Morphine pretreatment (100 mg/kg, s.c., -5 hr) produced antinociceptive tolerance as shown by a 2.7-fold increase in the calculated morphine A50 value. Tolerance was reversed by H7, but not H8, treatment (50 nmol, i.c.v., -30 min). These results are consistent with the hypothesis that a sensitized or constitutively active mu* state plays a role in narcotic tolerance and dependence.

Upon differentiation with retinoic acid of the human neuroblastoma cells SH-SY5Y into mature neurons, opioid drugs become highly effective in suppressing prostaglandin E1 (50% inhibition)- and forskolin (70% inhibition)-stimulated adenylate cyclase activity, which was assessed by measuring cyclic AMP accumulation in intact cells. Whereas the SH-SY5Y cells carry both mu and delta receptors in a ratio of mu/delta approximately equal to 5/1, the response is predominantly mediated by the mu receptor. Morphine acts as a strong agonist with an EC50 of 50 to 100 nM which falls into the therapeutic range expected for narcotic analgesic effects mediated by the mu receptor. Narcotic analgesic drugs with only partial agonism fail to evoke full response, which suggests that this cell model could provide a rapid screening assay for narcotic analgesic efficacy. Continued exposure of the cells to morphine resulted in partial tolerance within 12 hr with a 4-fold shift of morphine's EC50 to higher concentrations, whereas longer morphine exposure did not cause any further shift. Thus, the differentiated SH-SY5Y cells provide a suitable system for studying the molecular mechanisms of the narcotic analgesics.

The validity of permeabilized cells as a model of DNA and RNA synthesis was studied with the use of mouse S-49 lymphoblastoma cells rendered permeable by exposure to L-alpha-lysophosphatidylcholine. The permeabilized cells readily incorporated exogenously supplied cytosine and uracil nucleotides into HClO4-insoluble macromolecular material. However, the incorporation of these tracers did not require the three other complementary nucleotides, and adenine, guanine or thymine nucleotide tracers were incorporated at much lower rates. These results, which were also obtained with permeabilized Abelsohn-leukaemia-virus-transformed mouse macrophages, mouse neuroblastoma cells and S-49 lymphoblastoma homogenates, are inconsistent with semi-conservative DNA replication or RNA transcription; rather, they suggest the involvement of terminal nucleotidyltransferase(s) that mediate the incorporation of uracil and cytosine nucleotides. DNA synthesis was restored when permeabilized cells or cellular homogenates were supplemented with denatured salmon testes DNA. These results suggest that endogenous cellular DNA is impaired in its function as a template for DNA replication and transcription in vitro. Metabolic channelling or compartmentation of nucleic-acid-precursor pathways could not be demonstrated in the permeabilized cells.

Opiate receptor sites in the rat brain were defined in vivo by measuring the binding of etorphine, sufentanil, diprenorphine, and naloxone in saturation and cross-competition experiments. The binding data were analyzed simultaneously, using a computerized curve-fitting technique with an extended least-squares nonlinear regression program. Three types of binding sites could be distinguished: site 1 (18 pmoles/g of brain), site 2 (15 pmoles/g of brain), and site 3 (20 pmoles/g of brain). Site 1 is bound selectively by sufentanil (the ratio of the apparent equilibrium dissociation constants K2/K1 approximately equal to 1200), etorphine (K2/K1 approximately equal to 20), and naloxone (K2/K1 approximately equal to 15), and it resembles the mu binding site previously demonstrated in vitro. Diprenorphine binds to both site 1 and site 2 with high affinity and a slight (approximately 3.7-fold) selectivity for site 1 over site 2. The latter site may represent a mixture of the delta and kappa binding sites. The third site displays relatively high affinity for naloxone, but it is clearly different from sites 1 and 2, as it exhibits a lack of affinity for sufentanil, etorphine, and diprenorphine. This binding site population does not resemble any of the known opiate binding sites. Recent in vitro binding studies revealed that site 3 (now termed lambda site) is highly labile in vitro and was, therefore, not previously detected. These results suggest significant differences between in vitro and in vivo opioid receptor binding characteristics.

Specific assay procedures were developed to measure plasma concentrations of (±)‐1,2‐bis(3,5‐dioxopiperazinyl) propane (I) by GLC using flame‐ionization detection with a sensitivity limit of 5 μg/ml and by GLC‐mass fragmentography with a sensitivity limit of 0.2 μg/ml. Applicability of the assay procedures was demonstrated in rats, rabbits, and humans. Plasma concentration‐time curves of total 14C activity and intact I were obtained in rats and rabbits following oral and intravenous administration of 14C‐I. Plasma elimination half‐lives of I in the first 2 hr following intravenous doses in rats were 40 and 45 min in two rats. Plasma levels of I were measured over 6 hr after an intravenous dose in rabbits and followed a two‐compartment open model with a terminal loglinear plasma half‐life of 85 min. Significantly higher total 14C levels compared to intact I plasma concentrations indicated rapid biotransformation in both rats and rabbits to unknown metabolites. The oral bioavailability appeared to be limited in both species relative to intravenous administration. Two patients receiving 3 g of I/m2in tablets orally showed plasma levels of I similar to those obtained after oral doses in rats and rabbits, with peak concentrations at 2 hr after the dose (3.8 μg/ml) and with still measurable levels 12 hr after the dose (0.4 μg/ml).

A series of neuroblastoma cell lines were screened for the presence of opioid receptor sites with the tracers [3H]diprenorphine (mu, delta, kappa ligand) and [3H]naloxone (mu-selective ligand). One human neuroblastoma cell line, SK-N-SH, displayed avid binding for both tracers. Binding experiments with multiple tracers revealed the presence of both mu and delta sites. These sites were stereospecific, saturable, and proteinaceous in character. Saturation binding experiments provided an estimate of 50,000 mu and 10,000 delta sites/cell. NaCl (100 mM) and guanine nucleotide, guanylyl imidodiphosphate (50 microM), reduced opioid agonist but not antagonist binding to these sites. Etorphine at 1 nM inhibited prostaglandin E1-stimulated cyclic AMP production by approximately 20%, which was reversible by naloxone. The opioid-binding sites on SK-N-SH cells closely resemble the previously reported mu and delta sites in human and rodent brain. Therefore, the SK-N-SH neuroblastoma cell line represents a useful tool to study the molecular functions of opioid receptors.

The disposition of morphine in rat brain and serum was determined over 48 hr after subcutaneous doses. Free morphine was measured by a specific assay using 3H-labeling together with high-pressure liquid chromatography separation, with a sensitivity of 1 nM (0.3 ng of morphine per ml). This study revealed the persistence of free morphine in nanomolar concentrations over at least 24 hr after a single analgesic dose. The terminal half-life of morphine elimination was 5 hours. Total radioactivity was retained in the body at much higher concentrations. Similar disposition of [C-1-3H]morphine and [N-14CH3] morphine ruled out any major metabolic alterations at these positions, including N-demethylation. Irreversible binding to insoluble tissue components, which has previously been linked to tolerance, was observed only to the extent of less than 20% of total tissue radioactivity and was not unique to brain tissue. The persistence of morphine and its metabolites may be related to protracted opiate effects such as withdrawal symptoms after addiction.