The binding of SCH 39166 and SCH 23390 to 5-HT1C receptors in porcine choroid plexus

SCH 39166 is a novel benzonaphthazepine, which has been characterized as a potent and selective D1 antagonist. Recently, its D1 selective benzazepine predecessor, SCH 23390, has been shown to bind to 5-HT1C binding sites in the choroid plexus. Therefore, the present studies were undertaken to determine if SCH 39166 has any measurable affinity for 5-HT1C binding sites. Our results indicate that SCH 39166 exhibited poor affinity for the 5-HT1C receptor, with a Ki of 1327 nM. In contrast, SCH 23390 inhibited [3H]-mesulergine binding to 5-HT1C receptors with a Ki of 30 nM. The non-selective 5-HT antagonist, methysergide, inhibited binding with a Ki of 2.4 nM. Finally, studies with the stereoisomers of SCH 39166 and SCH 23390 demonstrated that stereoselectivity at the 5-HT1C site is significantly less than for the D1 site.     

Pharmacological effects of a specific dopamine D-1 antagonist SCH 23390 in comparison with neuroleptics

Neuroleptics such as thioxanthenes (cis($\text{Z}$)-flupentixol and cis($\text{Z}$)-clopenthixol) and phenothiazines (fluphenazine and perphenazine), which block both dopamine (DA) D-1 and D-2 receptors and the butyrophenones (haloperidol and spiroperidol), which block D-2 receptors only, are equipotent both behaviorally and clinically. A new compound SCH 23390 which selectively blocks DA D-1 receptors, resembles many neuroleptics in its pharmacological profile: antistereotypic effects in mice, rats and dogs, cataleptogenic effect and inhibitory effect on amphetamine circling. In contrast SCH 23390 has no effect on apomorphine-induced vomiting in dogs and little effects on 6-OHDA-denervated supersensitive DA receptors, stimulated by the DA agonist 3-PPP. In a series of experiments where methylphenidate-induced stereotyped gnawing in mice was inhibited by neuroleptics, it was shown that concomitant treatment with scopolamine or diazepam attenuated the effect of butyrophenones (D-2 antagonists). The same treatment attenuated the effect of phenothiazines, to a lesser extent, and hardly attenuated the effect of thioxanthenes and SCH 23390 at all. It is concluded that DA D-1 receptors are as important as D-2 receptors for the expression of neuroleptic activity in most animal models believed to be predictive of antipsychotic and extrapyramidal side-effect potential. However, the D-1 antagonist is less sensitive than D-2 antagonists to antimuscarinic compounds and benzodiapines.     

Characterization of the radioiodinated analogue of SCH 23390: in vitro and in vivo D-1 dopamine receptor binding studies

A new radioiodinated molecule, 125I-SCH 38840 (previously referred to as 125I-SCH 23982), has been recently reported to be a D-1 dopamine receptor ligand. The current study confirms and expands the characterization of both the radiolabeled and unlabeled forms of this compound, as well as describing the development of an in vivo D-1 receptor binding assay utilizing the 125I-SCH 38840. The binding of 125I-SCH 38840 to rat striatal membranes, in vitro, was saturable and exhibited a KD of 1.47 nM. Competition studies using 125I-SCH 38840 exhibited a pharmacological profile consistent with the proposal that 125I-SCH 38840 was binding to the D-1 receptor. Further studies with the unlabeled SCH 38840 demonstrated that it inhibited dopamine-stimulated adenylate cyclase with a KI of 66.1 nM, indicating that SCH 38840 was acting as a D-1 antagonist. Behavioral studies demonstrated that SCH 38840 (MED = 1.0 mg/kg, s.c.) blocked conditioned avoidance responding in rats, a measurement considered predictive of anti-psychotic activity in man. In vivo binding of 125I-SCH 38840 to rat striatum following s.c. administration was specific. Peak striatal levels were observed 1 h after injection, with measurable binding observed out to 8 h post-treatment. The displacement of the in vivo binding by unlabeled standards again suggested a D-1 selective interaction. The half-life of the in vivo binding of 125I-SCH 38840 was approximately 1.25 h, and was nearly equivalent to the half-life of the anti-CAR activity of unlabeled SCH 38840. These results clearly demonstrate the D-1 nature of SCH 38840's behavioral activity and strengthen the anti-psychotic potential of a D-1 antagonist.     

D-2 dopamine antagonist-like effects of SCH 23390 on A9 and A10 dopamine neurons

The effects of SCH 23390 on d-amphetamine-induced suppression of A9 and A10 DA neuronal firing were determined. SCH 23390 potently reversed d-amphetamine on both A9 and A10 DA neurons. Compared to haloperidol, SCH 23390 was 5 times more potent on A9 DA neurons and 20 times more potent on A10 DA neurons. However, the magnitude of the reversal effect was greater with haloperidol than SCH 23390. In addition, haloperidol produced a further increase in firing of both A9 and A10 DA neurons after SCH 23390 maximally increased firing. It was concluded that SCH 23390 has D-2 DA antagonist-like properties, possibly mediated via an interaction at D-1 DA receptors, which may be functionally linked with D-2 DA receptors. The marked potency of SCH 23390 in reversing d-amphetamine could be due to its combined antagonist effects at 5HT2 and D-1 DA receptor sites.     

Effects of SCH23390 and raclopride on a run-climb-run behavioral task in rats

The present study was designed to compare the putative differential behavioral consequences of treatment with SCH23390 (a selective dopamine D1 receptor blocker) and raclopride (a selective dopamine D2 receptor blocker) by employing a run-climb-run (RCR) behavioral task of different lengths. Rats were trained to traverse an uncovered floor alleyway (150 cm), climb a vertical rope (70 or 130 cm), and run across an upper board (100 cm) to access water for the reinforcement. At doses of 0.05, 0.10 and 0.15 mg/kg administered intraperitoneally 60 min before the behavioral session, both SCH23390 and raclopride significantly increased the total time to complete the tasks in a dose-related fashion. Microstructural analysis on the RCR behavioral performance revealed that the most apparent impairment induced by either drug was observed as the subject shifted motion from the end of the floor alleyway to the rope when hopping or to initiate climbing. However, the motion shift from climbing to running on the upper board was significantly impaired by raclopride, but not by SCH23390. Surprisingly, neither SCH23390 nor raclopride affected the climbing response itself. Running responses on the floor alleyway board were significantly disrupted by raclopride, whereas those on the upper board were significantly disrupted by SCH23390. Deficits induced by both drugs were more profound for the longer compared to the shorter rope, and were most notably shown at the transition area from running to climbing. These data indicate that both dopamine D1 and D2 receptors are involved in the RCR behavior performance. The results also suggest that the cost of motoric demand for behavioral performance is important for evaluating of the effects of drugs blocking dopamine receptors.     

Lethal effects of cocaine are reduced by the dopamine-1 receptor antagonist SCH 23390 but not by haloperidol

The prevalence of cocaine abuse has been associated with a host of medical complications and deaths. We investigated the effects of two dopamine antagonists with different affinities for dopamine-1 and dopamine-2 receptor subtypes on cocaine-induced lethality. Male Fischer-344 rats were given cocaine HCl (i.p.) and observed for lethality at 24 hrs. Cocaine was not lethal at 50 mg/kg and produced a steep dose-effect function from 60 to 100 mg/kg. Lethality was 88.9% at 100 mg/kg and the LD 50 was 79.7 mg/kg (95% CL: 74.8-84.9). Doses as high as 180 mg/kg failed to kill all rats. Lethality was often but not invariably associated with convulsions. Haloperidol (0.3-3 mg/kg i.p.) given 30 min prior to cocaine did not alter the lethal effects of cocaine but did reduce the lethality of methamphetamine. SCH 23390 (0.1-1 mg/kg i.p., 30 min prior) shifted the cocaine dose-effect function to the right at 0.3 mg/kg. Maximum protection was conferred by 0.3 mg/kg SCH 23390 where the LD 50 was increased to 100.1 mg/kg (95% CL: 91.5-109.5). Comparable protection was not observed if SCH 23390 was given 5 min after cocaine. These results suggest that dopamine receptors may play a role in the lethal effects of cocaine and that the D1 dopamine receptor subtype appears to be more relevant to lethality than the D2 subtype.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Phosphorylation-dephosphorylation of muscarinic acetylcholine receptors: evidence for the in vivo and in vitro release of receptors from rat brain plasma membrane

The effects of phosphorylation on muscarinic acetylcholine receptors (mAChRs) were studied in vitro and in vivo using rat brain plasma membrane and receptors partially purified at least 2500-fold. Purified mAChRs were phosphorylated in vitro by cAMP-dependent protein kinase and dephosphorylated by calcineurin. Phosphorylation of purified mAChRs was enhanced by carbachol and blocked by atropine. The filtrate which passed through glass fiber filters and high speed supernates were assayed for mAChRs by an ammonium sulfate precipitation method. Following incubation of the plasma membrane under phosphorylating conditions and ultracentrifugation at 300,000 g, the mAChRs appeared in the high speed supernate. This release was stimulated by adding carbachol to the incubation medium. In rats treated with carbachol, brain mAChRs redistributed from the heavy into the light membrane fractions. Ultrastructural examination of the light membrane fractions and the 300,000 g supernatant fractions after in vivo and in vitro carbachol treatment calcineurin increased the reincorporation of added partially purified receptors into the plasma membrane. The release and reincorporation of mAChRs strongly imply that there is a translocation and recycling of mAChRs between plasma membrane and cytosol in vivo. The significance and the function of the translocation of mAChRs remain to be investigated.     

Effects of cocaine alone and in combination with haloperidol and SCH 23390 on cardiovascular function in squirrel monkeys

The potential involvement of D1 and D2 dopamine receptors in the effects of cocaine on cardiovascular function in squirrel monkeys was evaluated. A low dose of cocaine (0.1 mg/kg i.v.) produced increases in both blood pressure and heart rate. At the higher doses of cocaine (1.0-3.0 mg/kg) the heart rate response was biphasic, consisting of an early decrease followed by an increase in heart rate 10-20 min following injection. The dopamine D2 antagonist haloperidol (0.1 mg/kg i.m.) attenuated the heart rate increasing effect of cocaine, but doses as high as 0.03 mg/kg did not alter the blood pressure increase. The D1 antagonist SCH 23390 (0.01-0.03 mg/kg i.m.) did not attenuate either the blood pressure or heart rate increasing effects of cocaine. The D2 agonist quinpirole (1.0 mg/kg i.v.) produced increases in heart rate similar to cocaine, with little effect on blood pressure. Although effective against the heart rate increasing effect of cocaine, haloperidol (0.01 mg/kg) did not antagonize the heart rate increasing effects of quinpirole. The D1 agonist SKF 38393 (3.0 mg/kg i.v.) decreased heart rate and increased blood pressure. The blood pressure increasing effect of SKF 38393 was antagonized by 0.01 mg/kg SCH 23390. Haloperidol's ability to partially antagonize the tachycardiac response to cocaine suggests the involvement of D2 receptors in that response. However, the failure of haloperidol to antagonize quinpirole's tachycardiac effect suggests that non-dopaminergic mechanisms may also be involved in haloperidol's antagonism of cocaine's tachycardiac effect. The pressor effects of cocaine do not appear to be controlled by selective dopamine receptors.     

Nicotine trapping causes the persistent desensitization of alpha4beta2 nicotinic receptors expressed in oocytes

To determine whether prolonged nicotine exposure persistently inactivates rat alpha4beta2 nicotinic receptors expressed in Xenopus oocytes, we measured the voltage-clamped alpha4beta2 response to acetylcholine (ACh) before and 24 h after, 1-h or 12-h incubations in 10 microm nicotine. A 12-h incubation in 10 microm nicotine depressed the alpha4beta2 ACh response for 24 h without affecting total or surface alpha4beta2 expression. To determine whether oocyte-mediated nicotine release caused this depression, we co-incubated an alpha4beta2-expressing oocyte with an un-injected one (pre-incubated in 10 microm nicotine for 12 h) for 24 h and measured the change in the alpha4beta2 ACh response. The response decreased by the same factor after the co-incubation as it did after a 12-h incubation in 10 microm nicotine and a 24-h incubation in nicotine-free media. Thus, oocyte-mediated nicotine release caused the persistent desensitization we observed after a 12-h incubation in 10 microm nicotine. Consistent with this result, measurements of [3H]nicotine release show that oocytes release enough nicotine into the wash media to desensitize alpha4beta2 receptors and that prolonged incubation in 300 microm ACh (which cannot readily cross the membrane or accumulate in acidic vesicles) did not persistently depress the alpha4beta2 response.     

The D1 receptor antagonist, SCH 23390, induces signs of parkinsonism in African green monkeys.

Systemic administration of the selective D1 antagonist, SCH 23390, caused significant motor changes in healthy African green monkeys. The effects included the parkinsonian signs of motor freezing, incoordination, bradykinesia, poverty of movement, tremor and depressed blink rate. SCH 23390 administered to MPTP-treated monkeys increased existing parkinsonism. The results are of particular interest in light of recent data that demonstrate the effectiveness of dihydrexidine, a full D1 agonist, in alleviating parkinsonism in MPTP-treated monkeys. These data implicate D1 receptors in the functions impaired by Parkinson's disease and suggest the possibility of parkinsonian side effects in the clinical use of this or similar D1 antagonists as treatments for psychiatric disorders.     

Retroviral vectors for persistent expression in vivo.

Retroviral vectors provide a safe and efficient method of introducing genes of therapeutic interest into dividing cells. The principle limitation of these vectors in the past has been poor gene expression in vivo. This problem has been overcome recently through the use of tissue-specific enhancers in commonly used retroviral vectors. In this review we discuss both the relevant biology and some of the practical applications of retroviral vectors in gene therapy.     

The antisecretory effects of clozapine,a dopamine D4 receptor antagonist,are blocked by the dopamine D1 receptor antagonist,SCH23390

Dopaminergic compounds affect gastric secretion and response to experimental gastric mucosal injury. We showed previously that the novel dopamine D₄ receptor antagonist, clozapine, significantly reduces gastric acid secretion and restraint stress-induced gastric lesions. Because the selectivity of clozapine for D₄ receptors has recently been questioned, we tested the ability of a known d₁ receptor blocker, SCH23390, to affect clozapine-induced reduction in gastric acid secretion. SCH23390 given i.p. or i.c.v., at doses that did not affect gastric acid secretion, significantly blocked the anti-secretory effect of clozapine, administered either peripherally or centrally. These data suggest that neither clozapine nor SCH23390 exhibit as high a degree of selectivity for the dopamine D₄ and d₁ receptor, respectively, as previously believed.     

Relapse prevention in schizophrenia: evidence from longterm, randomized, double-blind clinical trials

Fourteen long-term (duration at least 26 months) double-blind, randomized studies comparing second-generation antipsychotics (SGAs) with placebo, conventional antipsychotics, or two or more SGAs, published after 2002 have been reviewed and are summarized in this chapter. Methodological problems and factors influencing results such as patient populations involved, outcome criteria, and dosages of evaluated drugs are discussed.     

Experimental evidence for a persistent spore bank in Sphagnum

Spore capsules of four Sphagnum species were buried at different depths in peat on a bog. Spore viability was determined after 0, 1, 2 and 3 yr. Viability generally declined with time, but viable spores were still found at all depths after 3 yr. The light-coloured spores of S. balticum and S. tenellum retained their viability better than the darker spores of S. fuscum and S. lindbergii . Survival was highest under wet but aerobic conditions, but was also high under humid or periodically desiccated conditions. By contrast, most spores stored under wet, anaerobic conditions died within 2–3 yr. These results, and predictions from them, are not consistent with earlier results for spores of long-lived and dominant bryophytes, or for seeds of phanerogams of undisturbed wetlands and forests. There was no correlation between spore size and longevity across species, but the small spores from small capsules of S. balticum and S. tenellum generally showed higher viability than those from the medium-sized and large capsules of the same species. This suggests a positive intraspecific relationship between longevity and dispersal distance. There was an indication of conditional dormancy, controlled by weather, in Sphagnum spores. The experiments indicate that Sphagnum spores can form a long-term persistent spore bank under suitable conditions, with a half-life of between 1 and 20 yr (mean across species of 2.6 and 5.0 yr at two depths studied), and with potential values in individual spore capsules of several decades, or even of centuries. Sphagnum spores kept refrigerated showed 15–35% viable spores after 13 yr. The capacity to form a persistent spore bank that can be activated whenever favourable conditions occur might help explain the wide geographical distribution of many Sphagnum species in the boreal and temperate zones, where they have managed to colonize almost every suitable patch of acidic, nutrient-poor wetland.     

Beta-adrenergic receptors: evidence for negative cooperativity.

The specific binding of [³H] (?)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled β-adrenergic receptors. Hill Plots of such binding data yield slopes (n_H=“Hill Coefficients”) less than 1.0, suggesting that negatively cooperative interactions among the β-adrenergic receptors may occur. The existence of such negative cooperativity was confirmed by a direct kinetic method. The dissociation of receptor bound [³H] (?)alprenolol was studied under two conditions: 1) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (?)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (?)alprenolol increases the rate of [³H] (?)alprenolol dissociation, indicating that negatively cooperative interactions among the β-adrenergic receptor binding sites do occur.     

Transient and selective overexpression of dopamine D2 receptors in the striatum causes persistent abnormalities in prefrontal cortex functioning

Increased activity of D2 receptors (D2Rs) in the striatum has been linked to the pathophysiology of schizophrenia. To determine directly the behavioral and physiological consequences of increased D2R function in the striatum, we generated mice with reversibly increased levels of D2Rs restricted to the striatum. D2 transgenic mice exhibit selective cognitive impairments in working memory tasks and behavioral flexibility without more general cognitive deficits. The deficit in the working memory task persists even after the transgene has been switched off, indicating that it results not from continued overexpression of D2Rs but from excess expression during development. To determine the effects that may mediate the observed cognitive deficits, we analyzed the prefrontal cortex, the brain structure mainly associated with working memory. We found that D2R overexpression in the striatum impacts dopamine levels, rates of dopamine turnover, and activation of D1 receptors in the prefrontal cortex, measures that are critical for working memory.     

Atrial natriuretic peptide clearance receptors in trout: effects of receptor inhibition in vivo.

Inactivation of circulating atrial natriuretic peptides (ANP) by specialized clearance (C) receptors has been characterized in mammals but has not been examined in fish. In the present study arterial blood pressure, urine flow, and urine electrolytes were measured in chronically cannulated rainbow trout, Oncorhynchus mykiss, during infusion of the specific C receptor inhibitor, SC-46542. C receptor inhibition decreased blood pressure and pulse pressure, increased heart rate and urine flow, but did not affect urinary electrolyte concentrations. These responses are consistent with those produced by exogenous ANP administration and indicate that: (1) trout possess C-type receptors capable of ANP inactivation, and (2) ANP-like molecules are continuously released and metabolized by trout in vivo. Phosphoramidon, an inhibitor of neutral endopeptidase, did not enhance the SC-46542 response, indicating that C receptors predominate in ANP inactivation in these fish.     

Melanopsin-dependent nonvisual responses: evidence for photopigment bistability in vivo

In mammals, nonvisual responses to light have been shown to involve intrinsically photosensitive retinal ganglion cells (ipRGC) that express melanopsin and that are modulated by input from both rods and cones. Recent in vitro evidence suggests that melanopsin possesses dual photosensory and photoisomerase functions, previously thought to be a unique feature of invertebrate rhabdomeric photopigments. In cultured cells that normally do not respond to light, heterologous expression of mammalian melanopsin confers light sensitivity that can be restored by prior stimulation with appropriate wavelengths. Using three different physiological and behavioral assays, we show that this in vitro property translates to in vivo, melanopsin-dependent nonvisual responses. We find that prestimulation with long-wavelength light not only restores but enhances single-unit responses of SCN neurons to 480-nm light, whereas the long-wavelength stimulus alone fails to elicit any response. Recordings in Opn4-/- mice confirm that melanopsin provides the main photosensory input to the SCN, and furthermore, demonstrate that melanopsin is required for response enhancement, because this capacity is abolished in the knockout mouse. The efficiency of the light-enhancement effect depends on wavelength, irradiance, and duration. Prior long-wavelength light exposure also enhances short-wavelength-induced phase shifts of locomotor activity and pupillary constriction, consistent with the expression of a photoisomerase-like function in nonvisual responses to light.