Dopamine receptor D2 regulates genes involved in germ cell movement and sperm motility in rat testes†.

The function of dopamine receptor D2 (D2R) is well associated with sperm motility; however, the physiological role of D2R present on testicular cells remains elusive. The aim of the present study is to delineate the function of testicular D2R. Serum dopamine levels were found to decrease with age, whereas testicular D2R expression increased. In rat testicular sections, D2R immunolabeling was observed in interstitial cells, spermatogonia, spermatocytes and mature elongated spermatids, whereas tyrosine hydroxylase immunolabeling was selectively detected in Leydig cells. In vitro seminiferous tubule culture following bromocriptine (D2R agonist) treatment resulted in decreased cAMP levels. Microarray identified 1077 differentially expressed genes (511 up-regulated, 566 down-regulated). The majority of differentially expressed genes were present in post-meiotic cells including early and late spermatids, and sperm. Gene ontology elucidated processes related to extra-cellular matrix to be enriched and was supported by differential expression of various collagens and laminins, thereby indicating a role of dopamine in extra-cellular matrix integrity and transport of spermatids across the seminiferous epithelium. Gene ontology and enrichment map also highlighted cell/sperm motility to be significantly enriched. Therefore, genes involved in sperm motility functions were further validated by RT-qPCR. Seven genes (Akap4, Ccnyl1, Iqcf1, Klc3, Prss55, Tbc1d21, Tl18) were significantly up-regulated, whereas four genes (Dnah1, Dnah5, Clxn, Fsip2) were significantly down-regulated by bromocriptine treatment. The bromocriptine-stimulated reduction in seminiferous tubule cyclic AMP and associated changes in spermatid gene expression suggests that dopamine regulates both spermatogenesis and spermiogenesis within the seminiferous epithelium, and spermatozoa motility following spermiation, as essential processes for fertility.

Human Wharton's jelly-derived mesenchymal stem cells prevent pregnancy loss in a rat by JAK/STAT-mediated immunomodulation.

AIM: Spontaneous abortion (SA) is a multiple-original syndrome with immune imbalance as one of its major risk factors. As Wharton's jelly-mesenchymal stem cells (WJ-MSCs) are considered to be able to prevent abortion, this study aims to explore the currently poorly understood underlining molecular signaling pathways and regulatory mechanisms of WJ-MSCs in pregnancy maintenance. METHODS: Abortion mode is established by subcutaneous injection of bromocriptine in rat on day 9 and abortion prevention is achieved by WJ-MSCs injection via tail vein. WJ-MSCs were cultured with/without the inhibitors of JAK/STAT or NF-κB. The uterus was collected on the 14th day of gestation and the rate of embryo absorption was calculated. The expression of Th1/Th2/Th3 cytokines in decidual, placental tissue, and peripheral blood was analyzed. RESULTS: WJ-MSCs treatment significantly reduced the abortion rate in bromocriptine-treated pregnancy such that it was not significantly different from a normal pregnancy. JAK/STAT inhibition abolished pregnancy preserving effects of WJ-MSCs but NF-κB inhibition did not. The levels of Th1-related cytokines and mRNA levels in the bromocriptine abortion model were significantly higher than the normal pregnancy group and ethanol control group, while levels of the Th2-related cytokines and mRNA levels significantly decreased. WJ-MSCs transfusion into the abortion model restored cytokine profiles such that they were not significantly different from the normal pregnancy group and ethanol control group. JAK/STAT inhibition of WJ-MSCs prevented their effect on cytokine and mRNA levels, but NF-κB inhibition did not. CONCLUSIONS: WJ-MSCs significantly lower the rate of embryo resorption of spontaneous abortion by reducing Th1-related cytokines while increasing Th2 and Th3-related cytokines in JAK/STAT-dependent manner.

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.

Repurposing of FDA-approved drugs against active site and potential allosteric drug-binding sites of COVID-19 main protease.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still has serious negative effects on health, social life, and economics. Recently, vaccines from various companies have been urgently approved to control SARS-CoV-2 infections. However, any specific antiviral drug has not been confirmed so far for regular treatment. An important target is the main protease (Mpro ), which plays a major role in replication of the virus. In this study, Gaussian and residue network models are employed to reveal two distinct potential allosteric sites on Mpro that can be evaluated as drug targets besides the active site. Then, Food and Drug Administration (FDA)-approved drugs are docked to three distinct sites with flexible docking using AutoDock Vina to identify potential drug candidates. Fourteen best molecule hits for the active site of Mpro are determined. Six of these also exhibit high docking scores for the potential allosteric regions. Full-atom molecular dynamics simulations with MM-GBSA method indicate that compounds docked to active and potential allosteric sites form stable interactions with high binding free energy (∆Gbind ) values. ∆Gbind values reach -52.06 kcal/mol for the active site, -51.08 kcal/mol for the potential allosteric site 1, and - 42.93 kcal/mol for the potential allosteric site 2. Energy decomposition calculations per residue elucidate key binding residues stabilizing the ligands that can further serve to design pharmacophores. This systematic and efficient computational analysis successfully determines ivermectine, diosmin, and selinexor currently subjected to clinical trials, and further proposes bromocriptine, elbasvir as Mpro inhibitor candidates to be evaluated against SARS-CoV-2 infections.

The regulatory effect of bromocriptine on cardiac hypertrophy by prolactin and D2 receptor modulation.

BACKGROUND: Bromocriptine, a dopamine agonist, used for the treatment of hyperprolactinemia, type 2 diabetes, ovarian hyper-stimulation syndrome, has also effects on the cardiac remodeling process, but the mechanism of action is unknown. The aim of this work was to determinate the effect during hypertrophic process through molecular mechanisms that include prolactin receptor (Prlr) and receptor of dopamine 2 (D2 r) expression. METHODS: We used a model of cardiac hypertrophy induced by an aortocaval fistula (ACF) surgery in rats. Protein concentrations of D2 r and Prlr were determined by western blotting. The treatment consisted in water (control), captopril (50 mg/kg/day), bromocriptine (3 mg/kg/day), and ACF group (n = 6 per group). RESULTS: Our results showed that bromocriptine treatment decreases the hypertrophy index. Treatment with bromocriptine increases the protein expression of Prlr and D2 r in the cardiac tissue of rats with cardiac hypertrophy. CONCLUSIONS: We concluded that bromocriptine has a protective effect on cardiac hypertrophy, and due to this effect, it may modulate the expression of Prlr and D2 r, which are involved in the development of cardiac hypertrophy.

Structure of a D2 dopamine receptor-G-protein complex in a lipid membrane.

The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease1 and antipsychotic drugs2. DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine3, leading to stimulation of Gi and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-Gi complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular Gi-binding site. The DRD2-Gi structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders.

OATP1A2 and OATP2B1 Are Interacting with Dopamine-Receptor Agonists and Antagonists.

Interaction with the dopaminergic system in the central nervous system is either therapeutically intended or it is a side effect. In both cases, dopamine-receptor agonists (DRA) like the ergoline derivative bromocriptine and dopamine-receptor antagonists (DRAn) like metoclopramide have to cross the blood-brain barrier (BBB). The organic anion transporting polypeptides (OATP) 1A2 and 2B1 are cellular uptake carriers for a variety of endogenous and xenobiotic compounds. As both transporters are expressed in endothelial cells of the BBB, the aim of the present study was to determine whether the DRA bromocriptine, cabergoline, and pergolide and the DRAn metoclopramide and domperidone are interacting with OATP1A2 and 2B1 and could therefore be candidate genes modifying wanted and unwanted effects of these drugs. Localization of both transporters in the brain was confirmed using LC-MS/MS and immunofluorescence stainings. For the functional studies, MDCKII cells stably expressing OATP1A2 or 2B1 were used. Initial interaction studies with the well-characterized transporter substrate estrone 3-sulfate revealed that all tested compounds except pergolide inhibit the transport function of both proteins with the most potent effect for bromocriptine (IC50 = 2.2 µM (OATP1A2) and IC50 = 2.5 µM (OATP2B1)). Further studies using the indirect competitive counterflow method identified bromocriptine, cabergoline, and domperidone as substrates of both transporters, whereas metoclopramide was only transported by OATP1A2. These findings were verified for domperidone by direct measurements using its tritium-labeled form as a tracer. Moreover, the transporter-mediated uptake of this compound was sensitive to the OATP1A2 and OATP2B1 inhibitor naringin. In conclusion, this study suggests that OATP1A2 and 2B1 may play a role in the uptake of DR agonists and antagonists into the brain.

Placental transfer of bromocriptine in an ex vivo human placental perfusion model.

Purpose: To determine the rate and extent of the maternal-fetal transplacental passage of bromocriptine (BCT) in the dually perfused human placental model. Methods: Twenty term placentas were included in an ex vivo human placental perfusion experiment with a closed-circuit model. At the start of the perfusion, BCT at the concentration of 10 or 100 ng/ml along with 100 µg/ml antipyrine which used as a positive marker were added to the maternal reservoir. Samples were collected for the measurements of BCT and markers of placental viability both from the maternal reservoir and fetal reservoir throughout the perfusion which lasted for 3 h. Determination of BCT was carried out with liquid chromatography-tandem mass spectrometry. Results: At the end of the study, the concentration in the fetal compartment was 0.82 ± 0.32 ng/ml in the low concentration group and 5.02 ± 0.97 ng/ml in the high concentration group with a fetal transfer rate of 6.13 ± 1.94% and 5.46 ± 0.87%, respectively. Conclusion: These data showed that only trace amount of BCT could transport across the human placenta in vitro which suggested that fetal exposure to maternally administered BCT may be insignificant. More additional studies are required to explore the safety of BCT administrated in pregnancy.

ANNIVERSARY REVIEW: 50 years since the discovery of bromocriptine.

Ergotism is the long-term ergot poisoning by ingestion of rye or other grains infected with the fungus Claviceps purpurea and more recently by excessive intake of ergot drugs. It has either neuropsychiatric or vascular manifestations. In the Middle Ages, the gangrenous poisoning was known as St. Anthony's fire, after the order of the Monks of St. Anthony who were particularly skilled at treating the condition. In 1917, Prof. Arthur Stoll returned home to Switzerland from Germany, to lead the development of a new pharmaceutical department at Sandoz Chemical Company. Stoll, using the special methods of extraction learned from his work with his mentor Willstetter, started his industrial research work with ergot. He succeeded in isolating, from the ergot of rye, ergotamine as an active principle of an old popular remedy for excessive post-partum bleeding. The success of this discovery occurred in 1918 and was translated into a pharmaceutical product in 1921 under the trade name Gynergen. In subsequent work, Stoll and his team were leaders in identifying the structure of the many other alkaloids and amines produced by Claviceps purpurea This was the cultural background and scientific foundation on which bromocriptine was discovered.

Structural Investigation of the Dopamine-2 Receptor Agonist Bromocriptine Binding to Dimeric D2HighR and D2LowR States.

The active (D2HighR) and inactive (D2LowR) states of dimeric dopamine D2 receptor (D2R) models were investigated to clarify the binding mechanisms of the dopamine agonist bromocriptine, using Molecular Dynamics (MD) simulation. The aim of this comprehensive study was to investigate the critical effects of bromocriptine binding on each distinct receptor conformation. The different binding modes of the bromocriptine ligand in the active and inactive states have a significant effect on the conformational changes of the receptor. Based on the MM/GBSA approach, the calculated binding enthalpies of bromocriptine demonstrated selectivity toward the D2HighR active state. There is good agreement between the calculated and experimentally measured D2HighR selectivity. In the ligand-binding site, the key amino acids identified for D2HighR were Asp114(3.32) and Glu95(2.65), and for D2LowR, it was Ser193(5.42). Moreover, analysis of replicate MD trajectories demonstrated that the bromocriptine structure was more rigid at the D2HighR state and more flexible at the D2LowR state. However, the side chains of the ligand-receptor complex of D2HighR showed larger variations relative to the corresponding regions of D2LowR. The present study is part of an ongoing research program to study D2R conformational changes during ligand activation and to evaluate the conformational state selectivity for ligand binding.

An exploratory evaluation of tyrosine hydroxylase inhibition in planaria as a model for parkinsonism.

Planaria are the simplest organisms with bilateral symmetry and a central nervous system (CNS) with cephalization; therefore, they could be useful as model organisms to investigate mechanistic aspects of parkinsonism and to screen potential therapeutic agents. Taking advantage of the organism's anti-tropism towards light, we measured a significantly reduced locomotor velocity in planaria after exposure to 3-iodo-L-tyrosine, an inhibitor of tyrosine hydroxylase that is an enzyme catalyzing the first and rate-limiting step in the biosynthesis of catecholamines. A simple semi-automatic assay using videotaped experiments and subsequent evaluation by tracking software was also implemented to increase throughput. The dopaminergic regulation of locomotor velocity was confirmed by bromocriptine, a drug whose mechanisms of action to treat Parkinson's disease is believed to be through the stimulation of nerves that control movement.

Bromocriptine loaded chitosan nanoparticles intended for direct nose to brain delivery: pharmacodynamic, pharmacokinetic and scintigraphy study in mice model.

The primary aim of this study was to investigate the potential use of chitosan nanoparticles as a delivery system to enhance the brain targeting efficiency of bromocriptine (BRC) following intranasal (i.n.) administration. The BRC loaded chitosan nanoparticles (CS NPs) were prepared by ionic gelation of CS with tripolyphosphate anions. These NPs had a mean size (161.3 ± 4. 7 nm), zeta potential (+40.3 ± 2.7 mV), loading capacity (37.8% ± 1.8%) and entrapment efficiency (84.2% ± 3.5%). The oral administration of haloperidol (2mg/kg) to mice produced typical Parkinson (PD) symptoms. Catalepsy and akinesia outcomes in animals receiving BRC either in solution or within CS NPs showed a reversal in catalepsy and akinesia behavior when compared to haloperidol treated mice, this reversal being specially pronounced in mice receiving BRC loaded CS NPs. Biodistribution of BRC formulations in the brain and blood of mice following i.n. and intravenous (i.v.) administration was performed using optimized technetium labeled (99mTc-labeled) BRC formulations. The brain/blood ratio of 0.47 ± 0.04, 0.69 ± 0.031, and 0.05 ± 0.01 for BRC solution (i.n.), BRC loaded CS NPs (i.n.) and (i.v.) respectively, at 0.5h are suggestive of direct nose to brain transport bypassing the blood-brain barrier. Gamma scintigraphy imaging of mice brain following i.v. and i.n. administrations were performed to determine the localization of drug in brain. The drug targeting index and direct transport percentage for BRC loaded CS NPs following i.n. route were 6.3 ± 0.8 and 84.2% ± 1.9%. These encouraging results confirmed the development of a novel non-invasive nose to brain delivery system of BRC for the treatment of PD.

Structural and mechanistic insights into the interaction of cytochrome P4503A4 with bromoergocryptine, a type I ligand.

Cytochrome P4503A4 (CYP3A4), a major human drug-metabolizing enzyme, is responsible for the oxidation and clearance of the majority of administered drugs. One of the CYP3A4 substrates is bromoergocryptine (BEC), a dopamine receptor agonist prescribed for the inhibition of prolactin secretion and treatment of Parkinson disease, type 2 diabetes, and several other pathological conditions. Here we present a 2.15 Å crystal structure of the CYP3A4-BEC complex in which the drug, a type I heme ligand, is bound in a productive mode. The manner of BEC binding is consistent with the in vivo metabolite analysis and identifies the 8' and 9' carbons of the proline ring as the primary sites of oxidation. The crystal structure predicts the importance of Arg(212) and Thr(224) for binding of the tripeptide and lysergic moieties of BEC, respectively, which we confirmed experimentally. Our data support a three-step BEC binding model according to which the drug binds first at a peripheral site without perturbing the heme spectrum and then translocates into the active site cavity, where formation of a hydrogen bond between Thr(224) and the N1 atom of the lysergic moiety is followed by a slower conformational readjustment of the tripeptide group modulated by Arg(212).

Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian cytochrome P450.

Resonance Raman (RR) spectroscopy is used to help define active site structural responses of nanodisc-incorporated CYP3A4 to the binding of three substrates: bromocriptine (BC), erythromycin (ERY), and testosterone (TST). We demonstrate that nanodisc-incorporated assemblies reveal much more well-defined active site RR spectroscopic responses as compared to those normally obtained with the conventional, detergent-stabilized, sampling strategies. While ERY and BC are known to bind to CYP3A4 with a 1:1 stoichiometry, only the BC induces a substantial conversion from low- to high-spin state, as clearly manifested in the RR spectra acquired herein. The third substrate, TST, displays significant homotropic interactions within CYP3A4, the active site binding up to 3 molecules of this substrate, with the functional properties varying in response to binding of individual substrate molecules. While such behavior seemingly suggests the possibility that each substrate binding event induces functionally important heme structural changes, up to this time spectroscopic evidence for such structural changes has not been available. The current RR spectroscopic studies show clearly that accommodation of different size substrates, and different loading of TST, do not significantly affect the structure of the substrate-bound ferric heme. However, it is here demonstrated that the nature and number of bound substrates do have an extraordinary influence on the conformation of bound exogenous ligands, such as CO or dioxygen and its reduced forms, implying an effective mechanism whereby substrate structure can impact reactivity of intermediates so as to influence function, as reflected in the diverse reactivity of this drug metabolizing cytochrome.

Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity.

The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W, and L211F/D214E was studied with fluorescence resonance energy transfer and absorbance spectroscopy. Although in the wild type enzyme, the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (n(H)) around 1.7 (S(50) = 11.7 µM), the mutants showed no cooperativity (n(H) ≤ 1.1) with unchanged S(50) values. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high-affinity interaction is characterized by a dissociation constant (K(D)) ≤ 1.0 µM, whereas the K(D) of the second binding has the same magnitude as the S(50). Theoretical analysis of a two-step binding model suggests that n(H) values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event. Alteration of cooperativity in the mutants is caused by a partial displacement of the "spin-shifting" step. Although in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift on binding of the first substrate molecule.

Identification of neuroprotective compounds of caenorhabditis elegans dopaminergic neurons against 6-OHDA.

Parkinson's disease (PD) is a severe debilitating disorder, characterized by progressive and selective dopaminergic (DAergic) neuron degeneration within the substantia nigra pars compacta. Although current pharmacological treatments are effective in early stages of the disease, with time, most patients fail to respond to medications and develop serious motor complications. Therefore, devising novel and efficacious therapeutics that address not only the symptoms of PD, but also the cause, are of great importance. Unfortunately, many obstacles are associated with current PD research in mammalian-based systems, which limit the rate of progress. One solution is to investigate mechanisms of PD in model genetic organisms like Caenorhabditis elegans. In general, striking and profound similarities underlie the basic cellular and molecular processes between the worm and humans. The use of C. elegans over traditional mammalian-based systems holds the promise of an enhanced rate of discovery with lower associated costs. Here, we have utilized C. elegans to screen a variety of compounds, including specific dopamine (DA), GABA, and NMDA receptor agonists, as well as antagonists to identify those that protect against 6-OHDA-induced DAergic toxicity. Two DA D2 receptor agonists, bromocriptine and quinpirole, were found to protect against 6-OHDA toxicity in a dose-dependent manner. Surprisingly, these protective effects appear to involve receptor-independent mechanisms. Given the high degree of conservation of cellular processes between the worm and mammalian systems, these results are likely relevant and important toward understanding potentially novel mechanisms leading to DAergic neuroprotection in mammalian systems and, ultimately, new therapeutics for PD.

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W.

The contribution of conformational heterogeneity to cooperativity in cytochrome P450 3A4 was investigated using the mutant L211F/D214E/F304W. Initial spectral studies revealed a loss of cooperativity of the 1-pyrenebutanol (1-PB) induced spin shift (S(50)=5.4 microM, n=1.0) but retained cooperativity of alpha-naphthoflavone binding. Continuous variation (Job's titration) experiments showed the existence of two pools of enzyme with different 1-PB binding characteristics. Monitoring of 1-PB binding by fluorescence resonance energy transfer from the substrate to the heme confirmed that the high-affinity site (K(D)=0.3 microM) is retained in at least some fraction of the enzyme, although cooperativity is masked. Removal of apoprotein on a second column increased the high-spin content and restored cooperativity of 1-PB binding and of progesterone and testosterone 6beta-hydroxylation. The loss of cooperativity in the mutant is, therefore, mediated by the interaction of holo- and apo-P450 in mixed oligomers.

[Ropinirole in the treatment of Parkinson's disease: an update]. / Ropinirol en el tratamiento de la enfermedad de Parkinson: actualización.

INTRODUCTION: Ropinirole is a non-ergot dopaminergic agonist with a high affinity for D2 dopaminergic receptors which improves the symptoms of Parkinson's disease (PD) and delays the appearance of motor complications. It is different to the first generation of dopaminergic agonists in that, because it lacks an ergolinic structure, it does not have the side effects that usually appear with the use of this pharmacological group. DEVELOPMENT: Recent functional neuroimaging studies suggest a possible neuroprotector effect of the drug, although this aspect is still under discussion. The question as to when and how early treatment of PD must be started has been a controversial issue for many years now. Dopaminergic agonists have been used in monotherapy in patients with de novo disease with the intention of deferring treatment with levodopa and, in consequence, postponing the onset of the complications stemming from its use. Ropinirole has been evaluated in different studies both in monotherapy and as adjunctive therapy with levodopa. CONCLUSIONS: In the numerous clinical trials that were carried out, it would seem clear that ropinirole can be administered for years as sole early treatment for PD and that it offers a notable reduction in the appearance of dyskinesias. Given the linear dose-response relation it presents, the drug has a wide "therapeutic window" that allows the dosage to be increased as the disease progresses.

Usefulness of the thyrotropin-releasing hormone test in pre-clinical acromegaly.

Acromegaly is caused primarily by pituitary growth hormone (GH)-secreting tumors. It is usually recognized because of characteristic manifestations, and diagnosed clinically. However, there exists a mild stage of acromegaly, which poses a diagnostic problem due to the absence of typical clinical manifestations. Here we present four patients with pre-clinical acromegaly, who showed minimal acromegaloid features with elevated levels of insulin-like growth factor-I. Basal GH levels were within normal levels in 3 of 4 cases, while insulin-like growth factor-I levels were elevated above normal in all cases. Plasma GH levels were elevated in response to thyrotropin-releasing hormone (TRH) in all cases, indicating a diagnostic value of the TRH stimulation test. In contrast, an oral glucose tolerance test was not useful for the diagnosis, because of the low GH levels (less than 1 ng/ml) and/or secondary to diabetes mellitus. In response to a dopamine agonist, GH levels were increased in the two cases, whereas GH levels were decreased or remained unchanged in the other two cases. We therefore suggest that the TRH stimulation test would be helpful to examine the presence of pre-clinical acromegaly. Diagnosis of the early stages of acromegaly is important to prevent progression to overt acromegaly.

Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy.

We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.