The Inhibitory Effect of Selected D2 Dopaminergic Receptor Agonists on VEGF-Dependent Neovascularization in Zebrafish Larvae: Potential New Therapy in Ophthalmic Diseases.

Pathological angiogenesis is correlated with many ophthalmic diseases. The most common are exudative age-related macular degeneration and proliferative diabetic retinopathy. The current treatment for these diseases is based on regularly administered anti-VEGF antibodies injections. In the study, we investigated selected D2 dopaminergic receptor agonists, namely bromocriptine, cabergoline and pergolide, on hypoxia-induced neovascularization. We used the zebrafish laboratory model, specifically three-day post fertilization (dpf) Tg(fli-1: EGFP) zebrafish larvae. To induce abnormal angiogenesis of hyaloid-retinal vessels (HRVs) and intersegmental vessels (ISVs), the larvae were treated with cobalt chloride (II) (CoCl2) (a hypoxia-inducing agent) from 24 h post fertilization. The inhibitory role of D2 dopaminergic receptor agonists was investigated using confocal microscopy and qPCR. Additionally, the results were compared to those obtained in the group treated with CoCl2 followed by bevacizumab, the well-known antiangiogenic agent. Confocal microscopy analyses revealed severe deformation of vessels in the CoCl2 treated group, while co-incubation with bromocriptine, cabergoline, pergolide and bevacizumab, respectively, significantly inhibited abnormalities of angiogenesis. The qPCR analyses supported the protective role of the chosen dopaminergic agonists by demonstrating their influence on CoCl2-derived upregulation of vegfaa expression. The present results suggest that the D2 receptor agonists can be considered as a new direction in research for antiangiogenic therapy.

Pergolide, terguride and N,N'-spacer-linked oligomers of both interact with 5-HT2A receptors of rat tail artery.

Pergolide, terguride and N,N'-spacer-linked oligomers of both have been tested for their ability to interact with 5 hydroxytryptamine(HT)2A receptors of rat tail artery. Pergolide was a potent partial agonist (pEC50 7.5, Emax 55 %) and antagonized 5-HT-induced contractions (pKp 7.2). Pergolide dimer 3 with a p-xylene spacer between the indole nitrogens (N-1) displayed somewhat lower agonist potency than pergolide (pEC50 7.0, Emax 55 %, pKp 6.6). The contractile responses to pergolide and dimer 3 were antagonized by the 5-HT2A receptor antagonist ketanserin (pA2 9.4, 9.1). In contrast to pergolide dimer 3, pergolide dimers 5 and 9 with an alkyl and an aralkyl spacer between the piperidine nitrogens (N-6) lacked agonism and displayed low affinity at 5-HT2A receptors (pA2 < 5.5). Terguride behaved as an insurmountable antagonist of 5-HT (pA2 8.4). Oligomers of terguride showed 5 to 50-fold lower affinity. It is concluded that pergolide and terguride show a high affinity for 5-HT2A receptors, but dimerization (oligomerization) of both drugs fails to increase affinity.

Pergolide binds tightly to dopamine D2 short receptors and induces receptor sequestration.

Pergolide is an ergotamine derivative with potent D1 and D2 receptor activity. In this study we showed that pergolide binds tightly to dopamine D2 short receptors, as indicated by the long period of occupancy of the receptors after washing. Furthermore, pergolide induces receptor internalization to a larger extent than dopamine, seeing that no recycling of the receptors to the plasma membrane was observed for either agonist. The dissociation of pergolide from dopamine receptors occurs during the endocytotic process, leaving the receptors accessible to [3H]methylspiperone. Pergolide is a lipophilic compound that can reach and compete with [3H]methylspiperone for binding to sequestered receptors. If internalized receptors are still a target for drug action, pergolide could be a suitable compound of therapeutic interest in cases where receptor sequestration could prevent dopamine efficacy, as in levodopa therapy.

Endogenous dopamine limits the binding of antipsychotic drugs to D3 receptors in the rat brain: a quantitative autoradiographic study.

[3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin was used as a radioligand for the autoradiographic measurements of dopamine D3 receptors in rat and human brain. Preincubation of the brain sections was necessary to obtain binding of the radioligand in the islands of Calleja and in the nucleus accumbens, but not in cerebellar lobules 9/10 of the rat. D3 receptors were also totally occluded in unwashed sections of the human striatum. The radioligand binding to D3 receptors was maximal after preincubating the sections for at least 10 min. Pretreatment of the animals with reserpine or tetrabenazine, which results in a severe depletion of endogeneous monoamines, strongly reduces the occlusion of D3 receptors in unwashed brain sections. The occlusion of dopamine D3 receptors in brain sections suggests that the in vivo access to D3 receptors may be locally inhibited by endogenous dopamine. The in vitro binding affinities of 12 antipsychotic drugs for D2 and D3 receptors were evaluated in competition binding experiments, using both rat and cloned human receptors. Most of the compounds showed only a slightly lower affinity for D3 than for D2 receptors in vitro. Affinities of the antipsychotic drugs for cloned human D21 and D3 receptors were very close to their affinities for the rat receptors. In vivo occupancy of these receptors in the rat brain was measured ex vivo by quantitative autoradiography, 2 hours after subcutaneous drug administration. For most compounds, occupancy of D3 receptors, as compared to D2 receptor occupancy, was lower than expected from the corresponding in vivo affinity ratios. For the new antipsychotic risperidone, in vivo occupancy of D3 receptors was measured both in the islands of Calleja and in the cerebellar lobules 9/10. This compound was three times less potent for the occupancy of D3 receptors in the islands of Calleja than in the cerebellum, an area lacking endogenous dopamine (ED50 = 28 and 10 mg kg-1, respectively). Based on the observations in the rat brain, it may reasonably be supposed that therapeutic dosages of antipsychotic drugs will induce in patients only a minor occupancy of D3 receptors in brain areas containing high dopamine concentrations. The role of dopamine D3 receptors as a target of antipsychotic drugs may therefore be less important than previously thought.

Gpp(NH)p stimulates [3H]raclopride binding to homogenates from human putamen and accumbens.

The effects of the stable GTP analogue Gpp(NH)p (5'-guanylyl imido diphosphate) were examined on in vitro [3H]raclopride binding to dopamine D2 receptors in preparations from post mortem human brains. The estimated number of receptors in the brain was 29% and 38% higher in putamen and accumbens, respectively, when determined in the presence of Gpp(NH)p as compared to its absence. The interaction of agonists was biphasic confirming the two affinity state model of the receptor--G-protein complex. The addition of Gpp(NH)p to the assay abolished the two site competition of apomorphine with [3H]raclopride binding in both regions studied. The non-specific binding at high concentrations of apomorphine was not significantly affected by the addition of Gpp(NH)p, indicating that only the specific binding of [3H]raclopride to the dopamine D2 receptor is increased.

Synthesis and biodistribution of [125I]iodo- and [75Se]seleno-ergoline derivatives.

(8 beta)-8-([125]Iodomethyl)-6-propylergoline (125I-3) was prepared by refluxing the mesyl analog with Na125I in methyl-ethyl-ketone, followed by HPLC, in a radiochemical yield greater than 70%. [75Se]Selenopergolide (75Se-2) was prepared in 74% yield starting with H75(2) SeO3. The biodistribution studies of the two compounds in male rats show good uptake by the adrenals and the brain. Compound 75Se-2 had higher adrenal uptake and adrenal-to-blood ratios (4.2% dose/g and 70:1) than 125I-3 (3.6% dose/g and 23.8:1) at 15 min post injection. The two compounds had almost equal brain uptake (0.91% dose/g for 75Se-2 and 1.14% dose/g for 125I-3), but 75Se-2 showed higher brain-to-blood ratios (15.2:1 vs 7.3:1) at 15 min post injection. This study indicates that 75Se-2 and 123I-3 may be useful agents for imaging the adrenal and the brain.