Production and characterization of a specific 5-HT2 receptor antibody.

A synthetic peptide was used to generate antibodies against the rat serotonin-2 (5-HT2) receptor. The peptide corresponds to a unique sequence from the N-terminal extracellular portion of the receptor protein (antibody = Ab 5HT2-N). This peptide was chosen based on its theoretical antigenic index and for specificity to the 5-HT2 receptor. In dot blot analysis, antisera detected 2 ng-2 micrograms of synthetic peptide at dilutions of 1/200-1/20,000. COS-7 cells transiently transfected with a eukaryotic expression vector containing the 5-HT2 cDNA displayed intense immunoreactivity with crude and affinity-purified Ab 5HT2-N. In contrast, no immunoreactivity was seen in control experiments when: (1) non-transfected or vector transfected COS-7 cells were used; (2) pre-immune sera was substituted for primary antisera; (3) primary antisera was omitted; or (4) antiserum was pre-adsorbed to 10 microM synthetic peptide. Immunohistochemical analysis of sections of perfused rat brain revealed intense immunolabelling of a subset of neurons in regions of the ventral forebrain, dorsal hippocampus, striatum, cerebral cortex, and laterodorsal tegmental nucleus (LDT). An especially dense band of small cells was seen in layer 2 of pyriform cortex. There was a very high concentration of labelled cells in the laterodorsal tegmental nucleus. In situ hybridization histochemistry with a 5-HT2 antisense cRNA riboprobe showed a pattern of hybridization in forebrain similar to the pattern of immunolabelling with Ab 5HT2-N. Western blot analysis of proteins extracted from the LDT revealed a single protein species reacting with the antibody. This reactivity is not present in the pre-immune sera and is blocked by the synthetic antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin and the mammalian circadian system: I. In vitro phase shifts by serotonergic agonists and antagonists.

The primary mammalian circadian clock, located in the suprachiasmatic nuclei (SCN), receives a major input from the raphe nuclei. The role of this input is largely unknown, and is the focus of this research. The SCN clock survives in vitro, where it produces a 24-hr rhythm in spontaneous neuronal activity that is sustained for at least three cycles. The sensitivity of the SCN clock to drugs can therefore be tested in vitro by determining whether various compounds alter the phase of this rhythm. We have previously shown that the nonspecific serotonin (5-HT) agonist quipazine resets the SCN clock in vitro, inducing phase advances in the daytime and phase delays at night. These results suggest that the 5-HT-ergic input from the raphe nuclei can modulate the phase of the SCN circadian clock. In this study we began by using autoradiography to determine that the SCN contain abundant 5-HT1A and 5-HT1B receptors, very few 5-HT1C and 5-HT2 receptors, and no 5-HT3 receptors. Next we investigated the ability of 5-HT-ergic agonists and antagonists to reset the clock in vitro, in order to determine what type or types of 5-HT receptor(s) are functionally linked to the SCN clock. We began by providing further evidence of 5-HT-ergic effects in the SCN. We found that 5-HT mimicked the effects of quipazine, whereas the nonspecific 5-HT antagonist metergoline blocked these effects, in both the day and night. Next we found that the 5-HT1A agonist 8-OH-DPAT, and to a lesser extent the 5-HT1A-1B agonist RU 24969, mimicked the effects of quipazine during the subjective daytime, whereas the 5-HT1A antagonist NAN-190 blocked quipazine's effects. None of the other specific agonists or antagonists we tried induced similar effects. This suggests that quipazine acts on 5-HT1A receptors in the daytime to advance the SCN clock. None of the specific agents we tried were able either to mimic or to block the actions of 5-HT or quipazine at circadian time 15. Thus, we were unable to determine the type of 5-HT receptor involved in nighttime phase delays by quipazine or 5-HT. However, since the dose-response curves for quipazine during the day and night are virtually identical, we hypothesize that the nighttime 5-HT receptor is a 5-HT1-like receptor.

Serotonin and the mammalian circadian system: II. Phase-shifting rat behavioral rhythms with serotonergic agonists.

The suprachiasmatic nuclei (SCN) receive primary afferents from the median and dorsal raphe, but the role of these projections in circadian timekeeping is poorly understood. Studies of the SCN in vitro suggest that quipazine, a general serotonin (5-HT) receptor agonist, can produce circadian time-dependent phase advances and phase delays in circadian rhythms of neuronal activity. The present study addresses whether quipazine and the selective 5-HT1A receptor agonist 8-OH-DPAT are similarly effective in vivo. Drinking and wheel-running patterns of male Wistar rats individually housed in constant darkness were monitored before and after subcutaneous administration of quipazine (5-10 mg/kg) at either circadian time (CT) 6 or CT 18, with and without running wheels available. Dose-dependent phase advances (20-180 min) were produced at CT 6. Significant phase shifts were not observed at CT 18. CT 6 quipazine-treated animals also showed a sustained and significant shortening of rhythm period (tau) following treatment (-0.28 hr; p < 0.002). tau shortening was inconsistently observed in CT 18 quipazine-treated rats. Neither quipazine-induced phase shifts nor tau effects were dependent on wheel-running activity per se. 8-OH-DPAT delivered via intracerebral ventricular treatment into the third ventricle (5 microliters at 100 microM in saline) produced slightly smaller phase advances (20-90 min) at CT 6, but did not produce phase delays at CT 18 or changes in tau. These findings support in vitro evidence that 5-HT-ergic agonists can phase-shift the circadian pacemaker.

Glucocorticoid, interleukin-2, and prostaglandin interactions in a clonal human leukemic T-cell line.

We have studied the growth effects of conditioned media, interleukin-2 and PGE prostaglandin analogs on the glucocorticoid-sensitive human leukemic T-cell clone, CEM-C7. After 4 days, the glucocorticoid dexamethasone at approximately 10 nM kills 50% of CEM-C7 cells. To test the hypothesis that glucocorticoid-mediated lymphocytolysis was due to suppression of lymphokine expression only, we attempted to protect CEM-C7 cells from lysis by provision of lymphokine(s). Conditioned media from interleukin-2 secreting Jurkat T-cells as well as the glucocorticoid-insensitive, but receptor positive clone, CEM-C1, failed to prevent lymphocytolysis; exogenous interleukin-2 also did not provide protection. There were complex, biphasic interactions between dexamethasone and the synthetic PGEs, enisoprost and enisoprost free acid. Low doses of enisoprost alone (0.01 to 1 microgram/ml) stimulated growth, and in combinations completely reversed the growth inhibitory effects of 10 nM dexamethasone. Higher concentrations of enisoprost were inherently lethal and were additive to the steroid effect. Thus the glucocorticoid-induced lymphocytolysis in this human leukemic T-cell line may be modified biphasically by PGE prostaglandins, depending on their concentration. However, interleukin-2 or components in the conditioned media assayed had no effect in ameliorating the lethal response to glucocorticoid.

Pharmacokinetic and tolerance evaluation of actisomide, a new antiarrhythmic agent, in healthy volunteers.

The pharmacokinetics and tolerance of actisomide (SC-36602) were determined following intravenous doses of 2.1, 4.2, and 8.4 mg/kg infused over five hours. Plasma concentrations observed in the low-dose group (2.1 mg/kg) were below the assay's limit of detection and were not included in the pharmacokinetic analysis. The following pharmacokinetic parameters were obtained in the medium-dose (4.2 mg/kg) and high-dose (8.4 mg/kg) groups, respectively: peak plasma concentration 4.25 +/- 0.26 and 7.81 +/- 0.31 micrograms/mL; area under the plasma concentration versus time curve 19.79 +/- 2.96 and 39.81 +/- 7.05 h.micrograms/mL; elimination rate constant of the beta phase 0.105 +/- 0.77 and 0.093 +/- 0.009 h(-1), and half-life 8.85 +/- 4.61 and 7.51 +/- 0.69 h. Left ventricular ejection fraction decreased by 10, 11, and 16 percent in the low-, medium-, and high-dose groups, respectively. Heart rate was not altered during the low-dose infusion. At the medium- and high-dose levels, resting peak heart rate increased by 18 and 27 percent, respectively. Systolic and diastolic blood pressures were not significantly changed in any of the dose groups. Changes in electrocardiographic intervals for the three dose groups were not significant except at the highest dose where an average 20 percent increase in the QRS interval was seen. Mild subjective adverse effects (dizziness, taste perversion, and circumoral paresthesia) which did not necessitate discontinuing the infusion occurred in the highest dosage group. Further studies are warranted to more fully characterize the pharmacokinetic profile and therapeutic potential of actisomide.

Narcolepsy without unique MHC class II antigen association: studies in the canine model.

Human narcolepsy is almost exclusively associated with the major histocompatibility complex (MHC) class II antigen HLA-DR2 and is the strongest HLA-disease association described to date. Canine narcolepsy resembles the human disease in its behavioral manifestations and responses to therapeutic drugs. Therefore, mixed leukocyte culture (MLC) was used to study differences in the canine MHC class II (DLA-D) antigens present in narcoleptic dogs to determine whether an analogous, unique DLA-D antigen could be identified in canine narcolepsy. Results show at least five different DLA-D antigens appear in potential narcoleptic haplotypes among the 29 dogs studied. The data demonstrate that, unlike man, in dogs there is no unique D locus antigen associated with narcolepsy and further suggest that linkage disequilibrium with a specific MHC antigen is unlikely to be essential for the manifestation of canine narcolepsy. Because human narcolepsy is thought to be multigenic, the canine narcolepsy-MHC dissociation suggests that the dog model may help elucidate the non-MHC narcolepsy gene(s).

Hippocampal and cortical opioid receptor binding: changes related to the hibernation state.

In vitro opioid receptor binding in the dorsal hippocampal formation and parietal cortex was surveyed in ground squirrels (Citellus lateralis) in the contrasting physiological states of hibernation and euthermia (i.e. not hibernating). Computer-assisted autoradiographic analysis of coronal sections incubated with [3H]dihydromorphine (DMH; 4 nM) revealed statistically significant reductions in specific opioid binding associated with hibernation. In the dorsal hippocampal formation of hibernating animals, binding in the stratum radiatum of CA3, hilus of the dentate gyrus and molecular layer of the dentate gyrus exhibited decreases up to 34% compared to euthermic animals. The stratum radiatum of CA3 exhibited the smallest decrease overall. DHM binding in parietal cortex displayed significant hibernation-related reductions, although they were not uniformly observed across all laminae at the 3 different brain levels examined. These experiments present evidence of changes in brain opioid binding related to the mammalian state of hibernation. The results suggest that changes in opioid receptor binding during hibernation may contribute to the earlier reported apparent failure of morphine physical dependence to develop during hibernation.