Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist.

Family A G protein-coupled receptors (GPCRs) control diverse biological processes and are of great clinical relevance. Their archetype rhodopsin becomes naturally light sensitive by binding covalently to the photoswitchable tethered ligand (PTL) retinal. Other GPCRs, however, neither bind covalently to ligands nor are light sensitive. We sought to impart the logic of rhodopsin to light-insensitive Family A GPCRs in order to enable their remote control in a receptor-specific, cell-type-specific, and spatiotemporally precise manner. Dopamine receptors (DARs) are of particular interest for their roles in motor coordination, appetitive, and aversive behavior, as well as neuropsychiatric disorders such as Parkinson's disease, schizophrenia, mood disorders, and addiction. Using an azobenzene derivative of the well-known DAR ligand 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), we were able to rapidly, reversibly, and selectively block dopamine D1 and D2 receptors (D1R and D2R) when the PTL was conjugated to an engineered cysteine near the dopamine binding site. Depending on the site of tethering, the ligand behaved as either a photoswitchable tethered neutral antagonist or inverse agonist. Our results indicate that DARs can be chemically engineered for selective remote control by light and provide a template for precision control of Family A GPCRs.

Targeting G†Protein-Coupled Receptors by Capture Compound Mass Spectrometry: A Case Study with Sertindole.

Unbiased chemoproteomic profiling of small-molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein-coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure-activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most-active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.

Extremely low-frequency electromagnetic field exposure during chronic morphine treatment strengthens downregulation of dopamine D2 receptors in rat dorsal hippocampus after morphine withdrawal.

The aim of this study was to investigate the effect of extremely low-frequency electromagnetic field (ELF-EMF) exposure during morphine treatment on dopamine D2 receptor (D2R) density in the rat dorsal hippocampus following withdrawal. Rats were exposed to ELF-EMF (20 Hz, 14 mT) or sham exposed for 1h per day before injection of morphine (10mg/kg, i.p.) once daily for 12 days. The saline control group was sham exposed for the same period. Immunohistochemistry was used to detect the density of D2Rs on the 1st, 3rd and 5th morphine withdrawal days. The results showed that the density of D2Rs in sham-exposed morphine-treated rats on the 1st and 3rd days of morphine withdrawal was significantly lower than that of the saline control group. The ELF-EMF-exposed morphine group also exhibited a significantly lower density of D2Rs on the 1st and 3rd withdrawal days relative to the sham-exposed morphine group. However, the D2R density in both groups tended to recover as morphine withdrawal days increased. The results suggest that dorsal hippocampal D2Rs are sensitive to morphine withdrawal and that this is potentiated by ELF-EMF pre-exposure during morphine treatment.

Absence of circadian clock regulation of horizontal cell gap junctional coupling reveals two dopamine systems in the goldfish retina.

In fish and other vertebrate retinas, although dopamine release is regulated by both light and an endogenous circadian (24-hour) clock, light increases dopamine release to a greater extent than the clock. The clock increases dopamine release during the subjective day so that D2-like receptors are activated. It is not known, however, whether the retinal clock also activates D1 receptors, which display a much lower sensitivity to dopamine in intact tissue. Because activation of the D1 receptors on fish cone horizontal (H1) cells uncouples the gap junctions between the cells, we studied whether the clock regulates the extent of biocytin tracer coupling in the goldfish retina. Tracer coupling between H1 cells was extensive under dark-adapted conditions (low scotopic range) and similar in the subjective day, subjective night, day, and night. An average of approximately 180 cells were coupled in each dark-adapted condition. However, bright light stimulation or application of the D1 agonist SKF38393 (10 microM) dramatically reduced H1 cell coupling. The D2 agonist quinpirole (1 microM) or application of the D1 antagonist SCH23390 (10 microM) and/or the D2 antagonist spiperone (10 microM) had no effect on H1 cell coupling in dark-adapted retinas. These observations demonstrate that H1 cell gap junctional coupling and thus D1 receptor activity are not affected by endogenous dopamine under dark-adapted conditions. The results suggest that two different dopamine systems are present in the goldfish retina. One system is controlled by an endogenous clock that activates low threshold D2-like receptors in the day, whereas the second system is controlled by light and involves activation of higher threshold D1 receptors.

Energy-dependent UV light-induced disruption of (-)sulpiride antagonism of dopamine.

The dopamine D2 receptor antagonist sulpiride decreases the spontaneous locomotor activity of Planaria in an enantiomeric-selective and dose-dependent manner. We now report that (-)sulpiride (0.1 microM)-induced decrease of planarian locomotor activity is significantly (P<0.05) attenuated by low-energy (366 nm) ultraviolet (UV) light and to a greater extent by high-energy (254 nm) UV light. The phenomenon offers a novel approach for studying dopamine D2 receptor transduction processes in a simple in vivo model.

[An experimental study of the role of a blockade of serotonin 5-HT3 receptors and dopamine D2 receptors in the mechanism of the occurrence of early radiation vomiting in monkeys]. / Eksperimental'noe izuchenie roli blokady 5-HT3-retseptorov serotonina i D2-retseptorov dofamina v mekhanizme vozniknoveniia rannei radiatsionnoi rvoty u obez'ian.

The experiments with M. fasciculata monkeys exposed to 137Cs gamma-radiation with a dose of 6.9 Gy showed that Latranum, a blocker of serotonin 5-HT3 receptors, is a more efficient antiemetic than Dimetphramidum, a D2 dophamin lytic. This is suggested by fewer animals with emetic reaction of by less severe vomiting in case they have any. The results agree well with a hypothesis that serotonin receptors are dominant in the chemoreceptor trigger zone of monkeys.

Biological effects of prolonged exposure to ELF electromagnetic fields in rats: III. 50 Hz electromagnetic fields.

Groups of adult male Sprague Dawley rats (64 rats each) were exposed for 8 months to electromagnetic fields (EMF) of two different field strength combinations: 5microT - 1kV/m and 100microT - 5kV/m. A third group was sham exposed. Field exposure was 8 hrs/day for 5 days/week. Blood samples were collected for hematology determinations before the onset of exposure and at 12 week intervals. At sacrifice, liver, heart, mesenteric lymph nodes, bone marrow, and testes were collected for morphology and histology assessments, while the pineal gland and brain were collected for biochemical determinations. At both field strength combinations, no pathological changes were observed in animal growth rate, in morphology and histology of the collected tissue specimens (liver, heart, mesenteric lymph nodes, testes, bone marrow), and in serum chemistry. An increase in norepinephrine levels occurred in the pineal gland of rats exposed to the higher field strength. The major changes in the brain involved the opioid system in frontal cortex, parietal cortex, and hippocampus. From the present findings it may be hypothesized that EMF may cause alteration of some brain functions.

Prolonged exposure of chicks to light or darkness differentially affects the quinpirole-evoked suppression of serotonin N-acetyltransferase activity in chick retina: an impact on dopamine D4-like receptor.

Dopamine plays an important role in regulation of melatonin biosynthesis in retinas of several vertebrate species. In the avian retina, the dopamine receptor that controls melatonin production represents a D4-like subtype. Stimulation of this receptor by quinpirole (QNP) results in a dose-dependent decline of the nighttime activity of serotonin N-acetyltransferase (NAT, a key regulatory enzyme in melatonin biosynthesis) and melatonin level of the retina. The present study was undertaken to determine whether the ability of QNP to suppress nocturnal NAT activity of chick retina was affected by prolonged adaptation of animals to light and darkness. In the retina of chicks kept under a light:dark (LD) illumination cycle, dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels measured at the end of the light phase were significantly higher than those found in the middle of the dark phase. In animals maintained under continuous light (LL) or darkness (DD) dopamine and DOPAC contents of the retina measured at these two time points were similar and resembled levels found during, respectively, the light and dark phase in the retina of chicks kept under LD illumination cycle. Adaptation of chicks to LL and DD resulted in an attenuated and enhanced, respectively, response of the retinal NAT activity to the suppressive action of QNP. When compared to the LD group, a parallel shift to the right (LL group) or left (DD group) of the dose-response curve for QNP was observed, and the ED50 values for this dopamine receptor agonist were 3.4-times higher (LL) or 2.8-lower (DD) than those calculated for the control LD animals. It is suggested that prolonged exposure to light or darkness, by altering the level of the retinal dopaminergic neurotransmission, may modify the reactivity of the D4-like dopamine receptors regulating NAT activity of the chick retina.

Effects of neutron-gamma irradiation on striatal D1 and D2 receptor distribution.

The early effects of neutron irradiation on the striatal D1 and D2 dopaminergic receptor distribution were investigated by quantitative receptor autoradiography. One hour after exposure at the dose of 8.4 Gy, an increase of D1 (+21%) and D2 (+25%) receptor density was observed in the striatum, located at the most anterior levels, containing the richest plexus of dopaminergic fibers afferent from the substantia nigra. Regional differences in changes of D1 and D2 receptor density were observed. This up-regulation could contribute to the development of early radio-induced neuro-vegetative syndrome.

Early and transient effects of neutron irradiation on dopamine receptors in the adult rat brain.

The early neurochemical effects of neutron-gamma radiation exposures were studied through ligand dopamine D1, D2 receptors binding experiments. The parameters of binding were investigated on crude preparations from striatum at different delays (from 2 to 72 hours) after irradiation. An early and transient increase in the total number of sites was seen after exposure, even at infra-lethal dose. This 'radiosensitivity' was higher for D1 than for D2 receptor. It is assumed that these modifications could participate in the early neuro-vegetative syndrome observed in irradiated persons.

[The effect of ionizing radiation at superhigh doses on the processes of dopamine release and reuptake by nerve endings in different sections of the brain]. / Vliianie ioniziruiushchego izlucheniia v sverkhvysokikh dozakh na protsessy vysvobozhdeniia i obratnogo zakhvata dofamina nervnymi terminaliami razlichnykh otdelov golovnogo mozga.

Rats exposed to fast 24 MeV electrons (100 Gy) at the state of early transient incapacity (ETI) exhibited active release and reuptake of dopamine in nerve terminals of the striatum. No changes in the indices under study were found in rats exposed to 25 Gy radiation that did not cause the ETI development. The in vitro irradiation of the isolated synaptosomes (100 Gy) inhibited dopamine reuptake and increased the number of sites of 3H-spiperone binding to D2-receptors in a membrane fraction isolated from the striatum.