N-n-Propyl-substituted 3-(dimethylphenyl)piperidines display novel discriminative properties between dopamine receptor subtypes: synthesis and receptor binding studies.

3-Phenylpiperidines (PPEs) have been thoroughly investigated in view of their interesting dopaminergic activity, and the N-n-propyl substitution has been suggested as the most effective among several PPEs differently substituted on the phenyl ring. In previous studies, we found that the dimethyl substitution on the phenyl ring of N-unsubstituted PPEs provided compounds active toward alpha2-adrenergic receptors (alpha2-ARs), which proved to possess interesting selectivity properties. The high degree of homology between the binding domains of alpha2-ARs and D4-dopaminergic receptors (D4-DARs) prompted us to verify whether this kind of substitution on the aromatic ring might prove to be active against retinal DARs of the D4 subtype. On the basis of these premises, we synthesized the dimethylphenyl-substituted PPEs 4a-f, in which an n-propyl chain is present on the aminic nitrogen. Radioligand binding assays on bovine retina and striatum membranes for D1-like and D2-like DARs indicated that PPEs 4a, 4b, and 4f possess a high affinity and selectivity for the D4-DAR subtype of bovine retina.

Ro 15-4513, a partial inverse agonist for benzodiazepine recognition sites, has proconflict and proconvulsant effects in the rat.

The present report describes the effects of Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-(1,5-a) (1,4)-benzodiazepine-3-carboxylate) in the conflict test, on convulsions induced by isoniazid and DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) and on the binding of [3H]gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. Ro 15-4513 produced a dose-dependent proconflict effect that was prevented by the administration of the benzodiazepine antagonist, Ro 15-1788. In addition, Ro 15-4513 was not convulsant per se but enhanced the convulsions produced by isoniazid and completely blocked the convulsions induced by the full inverse agonist, DMCM. In vitro, Ro 15-4513, like ethyl-beta-carboline-3-carboxylate (beta CCE), antagonized the increase in [3H]GABA binding induced by diazepam. The results indicate that Ro 15-4513 is anxiogenic and interacts with benzodiazepine recognition sites as a partial inverse agonist.

Functional coupling of GABAA receptors and benzodiazepine recognition site subtypes in the spinal cord of the rat.

The interaction between GABAA receptors and benzodiazepine (BZD) recognition site subtypes in the spinal cord of the rat was investigated. Computer analysis of displacement curves for [3H]flunitrazepam [( 3H]FNT) binding by 2-oxo-quazepam (2OXOQ) indicated the presence of two subtypes of BZD recognition sites in this region. Type I sites accounted for approximately 25% of the total number of BZD recognition sites, the remainder being Type II sites. A similar proportion of Type I and Type II sites was obtained by Scatchard analysis of the saturation curves for [3H]FNT, [3H]2OXOQ and [3H]ethyl-beta-carboline-3-carboxylate [( 3H]beta CCE) binding. The in vitro addition of GABA (10(-8)-10(-4) M) to spinal cord membrane preparations produced an increase in the binding of [3H]FNT and [3H]2OXOQ. The maximal enhancement produced by GABA was 50 and 82% above control values for [3H]FNT and [3H]2OXOQ, respectively. In contrast, GABA stimulated both [3H]FNT and [3H]2OXOQ binding in the cerebellum to a similar extent. We also evaluated the effects of different ligands for BZD recognition sites on the binding of [3H]GABA to spinal cord membranes, as compared with brain areas containing a higher proportion ( greater than 30%) of Type I sites. Diazepam, quazepam and the beta-carboline, ZK 93423, enhanced the specific binding of [3H]GABA in a concentration-dependent manner (10(-7)-10(-5) M) in the cerebral cortex and hippocampus but not in the spinal cord and cerebellum. These results indicate that there is a regional variation in the interaction between GABA and BZD recognition sites in the central nervous system.

Decreased sensitivity to diazepam induced by chronic administration of FG 7142.

Chronic treatment with the beta-carboline inverse agonist FG 7142 (25 mg/kg i.p. twice a day for 15 consecutive days) enhances in rats the effects of proconvulsant and convulsant beta-carbolines and of the inverse agonist Ro 15-4513 whilst leaving unchanged the response to the benzodiazepine receptor antagonists. Moreover, the same treatment reduces the sedative and the anticonvulsant effects of diazepam. These results are consistent with the view that chronic treatment with FG 7142 may produce a decrease in the pharmacological effects of benzodiazepines, whilst inducing sensitization to the convulsant effect of inverse agonists.

Characterization of 3H-2-oxo-quazepam binding in the human brain.

1. 2-oxo-quazepam (2oxoquaz) is a novel benzodiazepine (BZD) hypnotic containing a trifluoethyl substituent on the ring nitrogen at position 1, which, unlike other BZDs, distinguishes two populations of BZD binding sites. In the present study we characterized the binding of 3H-2oxoquaz to human brain membrane preparations. 2. Self and cross displacement curves for 3H-FNT and 3H-2oxoquaz binding in different brain areas indicate that 2oxoquaz binds with different affinities to two populations of binding sites in the human brain. 3. Competition studies of 3H-2oxoquaz (2 nM) and 3H-FNT (0.5 nM) binding with a series of unlabelled ligands indicate that compounds which preferentially bind to Type I sites are more potent at displacing 3H-2oxoquaz than 3H-FNT from cerebral cortex membrane preparations. 4. The binding of 3H-2oxoquaz is stimulated by gamma-aminobutyric acid (GABA) and pentobarbital in a concentration-dependent manner. 5. The results suggest that in the human brain 3H-2oxoquaz binds with high affinity to a subpopulation of BZD recognition sites (Type I sites) which are functionally linked to the GABA receptor and the chloride ionophore.

Protective effects of the new lazaroid "U-83836E" in splanchnic artery occlusion (SAO) shock.

We studied the effects of the new antioxidant drug U-83836E during splanchnic artery occlusion (SAO) shock in the rat. Serum tumor necrosis factor (TNF-alpha), white blood cell (WBC) count, mean arterial blood pressure (MAP), survival rate and the responsiveness to acetylcholine of aortic rings were investigated. SAO shock produced a marked increase in serum TNF-alpha (241.4 +/- 18.2 U/ml vs Not Detectable in basal), reduced MAP (51.4 +/- 4 mmHg vs 85.1 +/- 5 mmHg), survival time (80 +/- 10 min vs > 240 min), WBC count (2.8 +/- 0.4 x 10(3)/mm3 cells vs 11.7 +/- 0.9 x 10(3)/mm3 cells) and blunted the responsiveness to ACh of aortic rings (60 +/- 3% tension vs 23 +/- 4% tension). The analogue of vitamin E, U-84836E, administered at onset of reperfusion, lowered serum TNF-alpha (38.4 +/- 6.5 U/ml; p < 0.001), improved MAP (67.5 +/- 3.8 mmHg; p < 0.001), WBC count (8.9 +/- 0.6 x 10(3)/mm3; p < 0.001), and survival time (235 +/- 15 min; p < 0.001), and restored the responsiveness to ACh of aortic rings (32 +/- 3.7% tension; p < 0.001). These preliminary data suggest that this new compound could be a promising drug in shock therapy.

Melatonin induces membrane conductance changes in isolated retinal rod receptor cells.

Experiments were conducted to verify whether the neurohormone melatonin influences the membrane conductance of photoreceptors isolated from the frog retina. It has been found that 20 microM melatonin decreases membrane conductances both in the linear and non linear ranges by <0.4 nS. These actions are estimated to produce in dark adapted photoreceptors an increase of the response to a dim light induced change of the dark current of about 21%, i.e. from 1.3 to 1.62 mV/pA.

Effects of melatonin on lipid peroxidation induced by oxygen radicals.

We here report the activity of the neurohormone melatonin (MLT) as a scavenger of free radicals in two different experimental models: (a) linoleic acid peroxidation initiated by different free radical-generating systems and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine. In system (a) linoleic acid peroxidation, induced by either the water-soluble initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (ABAP) or Fe2+-EDTA addition to 2.6 mM linoleic acid dispersed in SDS-phosphate buffer, was evaluated as the formation of conjugated dienes, measured spectrophotometrically at 236 nm. MLT did not reduce the rate of peroxidation induced by ABAP, but did reduce, in a concentration-dependent fashion, the rate of the reaction activated by Fe2+-EDTA. In system (b) multilamellar vesicles were used as the substrate for lipid peroxidation, initiated by Fe2+-EDTA and determined by means of malonaldehyde (MDA) and 4-hydroxyalkenal (4-HDA) content. MLT was found to be slightly more effective in system (b) than in the dispersed linoleic acid system (see a). These results show that MLT inhibits lipid damage induced by oxygen free radicals. However, MLT is only about one one-hundredth as effective an antioxidant as vitamin E in the micelles system.

Preferential affinity of 3H-2-oxo-quazepam for type I benzodiazepine recognition sites in the human brain.

The hypnotic drug quazepam and its active metabolite 2-oxo-quazepam (2-oxo-quaz) are two benzodiazepines (BZ) containing a trifluoroethyl moiety on the ring nitrogen at position 1, characterized by their preferential affinity for Type I BZ recognition sites. In the present study we characterized the binding of 3H-2-oxo-quaz in discrete areas of the human brain. Saturation analysis demonstrated specific and saturable binding of 3H-2-oxo-quaz to membrane preparations from human cerebellum. Hill plot analysis of displacement curves of 3H-flunitrazepam (3H-FNT) binding by 2-oxo-quaz yielded Hill coefficients of approximately 1 in the cerebellum and significantly less than 1 in the cerebral cortex, hippocampus, caudate nucleus, thalamus and pons. Self and cross displacement curves for 3H-FNT and 3H-2-oxo-quaz binding in these brain areas indicated that 2-oxo-quaz binds with different affinities to two populations of binding sites. High affinity binding sites were more abundant in the cerebellum (95% of total sites), cerebral cortex, hippocampus and thalamus, whereas low affinity sites were predominant in the caudate nucleus and pons. Competition studies of 3H-2-oxo-quaz (2 nM) and 3H-FNT (0.5 nM) using unlabelled ligands indicated that compounds which preferentially bind to Type I sites are more potent at displacing 3H-2-oxo-quaz than 3H-FNT from cerebral cortex membrane preparations. The results suggest that 3H-2-oxo-quaz may be used for selectively studying Type I BZ recognition sites in the human brain.

Dopamine agonists and analogues have an antiproliferative effect on CHO-K1 cells.

Epidemiological studies have shown a reduced incidence of cancer in Parkinson's disease. Since nearly all parkinsonian patients with clinical impairment are treated with L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA)ergic agonists, a possibility exists that these therapeutic agents can influence the risk of cancer. We studied the antiproliferative effect of these therapeutic agents (and substances structurally correlated) on Chinese hamster ovary (CHO)-K1 cell growth. Among the compounds tested, apomorphine proved to be the most potent inhibitor of CHO-K1 cell growth, with an EC(50) of 3.35 +/- 0.12 micro M. The apomorphine analogues, apocodeine and hydroxyethylnorapomorphine, were less active as inhibitors of CHO-K1 cell growth. The activity of DA, 6-hydroxydopamine (6-OHDA), phenylethylamine (PEA), L-DOPA and bromocriptine as antiproliferative was one order of magnitude lower than that of apomorphine while pergolide was ineffective. To test whether or not the oxidative potential of these compounds was important for their antiproliferative effect, several antioxidants were assayed. Among them glutathione (GSH) and dithiothreitol (DTT) were effective in reversing the anti-proliferative effect of apomorphine, DA, 6-OHDA and PEA, conversely they did not work with bromocriptine. GSH and DTT are sulphydryl-reducing agents; while their effect could explain the efficacy against apomorphine, DA and 6-OHDA, it is difficult to understand why they should have any effect on PEA as this substance does not react with sulphydryl groups. The oxidative potential as a mechanism of action was also questioned by the results obtained with dihydrorhodamine 123, a probe that changes its fluorescent emission wave when oxidized. None of the compounds, with the exception of 6-OHDA, had any effect on the fluorescent emission wave of the probe at the maximal concentrations used to inhibit CHO-K1 cell growth. At concentrations five times higher, apomorphine and DA generated reactive oxygen species but PEA and bromocriptine did not. These data demonstrate that the antiproliferative effect of these compounds is not due to their oxidative potential, but another mechanism must be postulated.

Cyclosporine-induced lipid peroxidation and propionyl carnitine protective effect.

Cell and tissue lipoperoxidation of the kidney induced by cyclosporine through the release of reactive oxygen species has recently been pointed out to be one of the factors responsible for the toxic phenomena related to the administration of cyclosporine. Our previous research on propionyl carnitine had shown an antilipoperoxidative effect of this substance on isolated cells such as erythrocytes and leukocytes, and also on the endothelial, vasal and cardiac tissues. In the experiments presented herein we also examined if propionyl carnitine could carry out its already well-known antilipoperoxidative effect in the renal tissue, and if this mechanism could be taken into consideration in order to explain the protective effect of propionyl carnitine against cyclosporine induced toxicity. Trials were carried out on isolated and perfused rat kidneys, and we were able to observe that propionyl carnitine exerted a protective action on toxic lipid peroxidation phenomena induced by cyclosporine. The results we obtained, together with other mechanisms which we had already proved regarding the intense protective activity of propionyl carnitine on cyclosporine-induced nephrotoxicity, complete the complex picture that describes the protective activity of propionyl carnitine against cyclosporine toxicity.

The energetic cost of photoreception in retinal rods of mammals.

The effects of temperature on rod sensitivity and adaptation are analysed in the general context of the energy requirements of photoreception. The dependence of adaptation on the [Na]i turn-over appears to be critical in mammalian rods where the metabolic load is particularly heavy because of both temperature conditions and large Na+ influx. Estimates of the energy dissipated by rods in darkness and during bright illumination show that the metabolic load is reasonably well distributed. From this analysis it also results that most of the energy, which a rod dissipates in both darkness and light, is needed to keep [Na]i and [Ca]i low.

Cell physiology of the pineal body.

The results from recent experiments on the cellular physiology of the trout pineal photoreceptors are briefly reviewed. The arguments are mainly concerned with pineal phototransduction. These studies have stimulated further research on melatonin, a molecule produced in pineal as well as in retinal photoreceptors. A discussion follows on our actual research object, that is a study of the influences of endogenous melatonin upon retinal receptor cells activities.

Melatonin as an antioxidant in retinal photoreceptors.

Dark-adapted, single photoreceptors isolated from the frog retina produce reactive oxygen species (ROS) after about 1 min of illumination with saturating light that we verified by their oxidation of preloaded dihydrorhodamine 123 (DHR) into the fluorescent rhodamine 123 (RHO). In this preparation we tested the antioxidant effects of vitamin E and of melatonin. Melatonin at picomolar and low nanomolar concentrations was determined to be 100 times more potent in inhibiting the light-induced oxidative processes than was vitamin E. On the contrary, both compounds exerted potent prooxidant effects at micromolar concentrations that is above the physiological levels of melatonin. This provides evidence that physiological concentrations of melatonin in a living cell may exert protective actions against a natural oxidant stimulus (light). This helps to define the functional role of endogenous melatonin in photoreceptors, which by their physiological characteristics, are among the marked producers of ROS in the organism.

Inhibition of lipid peroxidation by N-acetylserotonin and its role in retinal physiology.

N-Acetylserotonin (NAS) inhibits the peroxidation of linoleic acid induced by a water-soluble initiator, 2,2'-azobis(2-amidinopropane) (ABAP). Lipid peroxidation was detected by the formation of conjugated dienes, monitored spectrophotometrically at 236 nm. N-Acetylserotonin, at concentrations ranging from 200 nM to 20 microM, reduced the rate of ABAP-initiated lipid peroxidation in a concentration-dependent manner. The results of NAS-inhibited lipid peroxidation are compared to the antioxidant activities of melatonin, vitamin E, and a water-soluble vitamin E analog, Trolox. It is suggested that NAS acts as a physiological antioxidant in retinal photoreceptor cells.

Properties and functional roles of hyperpolarization-gated currents in guinea-pig retinal rods.

1. The inward rectification induced by membrane hyperpolarization was studied in adult guinea-pig rods by the perforated-patch-clamp technique. 2. CsCl blocked the rectification observed in both voltage- and current-clamp recordings at voltages negative to -60 mV, while BaCl2 blocked the inward relaxation observed at voltages positive to -60 mV. The current activated at -90 mV had a low selectivity between sodium and potassium and reversed at -31.0 mV. 3. These observations suggest that two inward rectifiers are present in guinea-pig rods: a hyperpolarization-activated (Ih) and a hyperpolarization-deactivated (Ikx) current. The functional roles of Ih and Ikx were evaluated by stimulating rods with currents sinusoidally modulated in time. 4. Rods behave like bandpass amplifiers, with a peak amplification of 1.5 at about 2 Hz. For hyperpolarizations that mainly gate Ikx, amplification and phase shifts are fully accounted for by a rod membrane analogue model that includes an inductance. For hyperpolarizations that also gate Ih, a harmonic distortion became apparent. 5. Bandpass filtering and amplification of rod signals, associated with Ih and Ikx gating by membrane hyperpolarization, are strategically located to extend, beyond the limits imposed by the slow phototransductive cascade, the temporal resolution of signals spreading to the rod synapse.

Retinal photoreceptors of Syrian hamsters undergo oxidative stress during streptozotocin-induced diabetes.

AIMS/HYPOTHESIS: The aim of this study was to verify whether retinal photoreceptors, like other tissues, are subject to oxidative stress during diabetes. METHODS: Oxidative stress was monitored by the oxidation of preloaded dehydrorhodamine 123 into fluorescent rhodamine 123, during a period of intense illumination of isolated rod retinal receptor cells. These were obtained from 22 Syrian hamsters injected with streptozotocin (50 mg/kg body weight., intraperitoneal route) 90 days before the study began. Eleven hamsters were treated daily with melatonin (0.4 mg/kg body wt., per os), an antioxidant synthesized within photoreceptors. Isolated photoreceptors were bathed on the stage of a Leitz Orthoplan microscope, where the fluorescent lamp also served as the light stimulus (485 nm). Fluorescence irradiance was measured by photometry and stored in a personal computer for further analysis. RESULTS: The light-induced oxidant production greatly decreased and was also delayed in the streptozotocin-injected hamsters compared with the control hamsters matched for age. Similar effects were obtained in control photoreceptors after 40 min incubation with 2-2'-azobis (2-amidinopropane) dihydrochloride, a potent lipoperoxidation inducer. The effect of melatonin was to partially restore the light-induced fluorescence response. CONCLUSION/INTERPRETATION: The depression of the light-induced oxidative response in diabetic photoreceptors could be ascribed to a hyperglycaemia-induced background of oxidative stress whereby the light-oxidizable substrate is actually lowered. Melatonin induces a larger fluorescence response during illumination, probably as a consequence of its antioxidant effect during diabetes, which would provide more oxidizable lipids.

Enhancement of gamma-aminobutyric acidA receptor function and binding by the volatile anesthetic halothane.

The volatile general anesthetics halothane and enflurane increased muscimol-stimulated 36Cl- efflux via gamma-aminobutyric acid (GABA)A receptors in rat brain cortical slices and also increased basal 36Cl- efflux in the absence of GABA agonist. The effects occurred in the clinical range of anesthetic concentrations (0.56-1.7 mM halothane and 0.46-1.4 mM enflurane). Both anesthetics induced a slow onset increase in basal 36Cl- efflux rate when added alone with no exogenous GABA agonist. This direct effect of halothane had a biphasic dependence on anesthetic concentrations, with a maximal effect in the range 1.1 to 1.7 mM. Replacing extracellular calcium with magnesium or blocking voltage-gated calcium entry with cobalt (200 microM) altered the direct halothane effect, shifting the concentration-dependence curve to the right. Halothane direct potentiation of chloride flux in the absence of GABA agonist was blocked by the GABAA chloride channel antagonist picrotoxin but not by the GABAA receptor antagonist bicuculline. The halothane potentiation of the muscimol response was detectable at concentrations of 0.56 mM halothane in the assay buffer, and was linear with concentration up to 2.8 mM. The effect was more pronounced at low GABA agonist concentrations, apparently due to an increase in GABA affinity. Lowering the extracellular calcium concentration to micromolar levels did not affect halothane potentiation of muscimol responses. Halothane at similar concentrations increased the high-affinity binding of [3H]muscimol to GABAA receptor sites in rat brain cortical membranes in a calcium-independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of gamma-aminobutyric acid binding by the anxiolytic beta-carbolines ZK 93423 and ZK 91296.

The effects of two anxiolytic beta-carboline derivatives, ZK 93423 and ZK 91296, on the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to brain membrane preparations from rat cerebral cortex were examined. ZK 93423 concentration-dependently enhanced the specific binding of [3H]GABA, with a maximal increase of 45% above control at a 50 microM concentration. A less pronounced increase was induced by diazepam and by the partial agonist ZK 91296. Scatchard plot analysis revealed that the effect of ZK 93423 was due to an increase in the total number of high- and low-affinity GABA binding sites. The action of ZK 93423 was mediated by benzodiazepine recognition sites since it was blocked by the benzodiazepine antagonists Ro 15-1788 and ZK 93426 at concentrations that failed to modify [3H]GABA binding on their own. Moreover the stimulatory effect of ZK 93423 on [3H]GABA binding was also blocked by the beta-carboline inverse agonist ethyl beta-carboline-3-carboxylate. These results are consistent with the view that ZK 93423 and ZK 91296, similarly to benzodiazepines, exert their pharmacological effects by enhancing the GABAergic transmission at the level of the GABA/benzodiazepine receptor complex.