Spiperone Stimulates Regeneration in Pulmonary Endothelium Damaged by Cigarette Smoke and Lipopolysaccharide.

BACKGROUND: Endothelial dysfunction and destruction of the pulmonary microcirculation are important pathogenic factors in chronic obstructive pulmonary disease (COPD). In COPD, bronchial obstruction is associated with endothelial dysfunction. Thus, new pharmacological treatment options aimed at restoring the pulmonary endothelium represent a clinical need in COPD therapy. Notch1 has been shown to protect cells against apoptosis, inflammation, and oxidative stress caused by cigarette smoke extract (CSE). Therefore, drug which effect on Notch1 may be a potential therapeutic target for COPD in the future. METHODS: In this study, we assessed the potential of spiperone to mediate regeneration of pulmonary endothelium in model of pulmonary emphysema induced by a CSE and lipopolysaccharide (LPS) in female C57BL/6 mice. RESULTS: Spiperone increased the number of capillaries as well as the expression of the CD31 in the alveolar tissue compared to the controls. Moreover, application of spiperone prevented alveolar wall destruction (DI), and reduced the area of emphysema. Lastly, we demonstrated that spiperone positively influenced mobilization and migration of endothelial progenitor cells (EPC, CD45-CD34+CD31+), CD309+-endothelial cells, and angiogenesis precursors (CD45-CD117+CD309+) into the lung. Spiperone administration significantly reduced the number Notch1 positive CD309+-endothelial cells and Notch1+ EPCs. CONCLUSION: Overall, our results suggest that spiperone mediates endothelial regeneration in an animal model of COPD. Thus, it could represent a novel therapeutic approach for treatment of emphysema associated with COPD.

Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level.

G protein-coupled receptors (GPCRs), including dopamine receptors, represent a group of important pharmacological targets. An increased formation of dopamine receptor D2 homodimers has been suggested to be associated with the pathophysiology of schizophrenia. Selective labeling and ligand-induced modulation of dimerization may therefore allow the investigation of the pathophysiological role of these dimers. Using TIRF microscopy at the single molecule level, transient formation of homodimers of dopamine receptors in the membrane of stably transfected CHO cells has been observed. The equilibrium between dimers and monomers was modulated by the binding of ligands; whereas antagonists showed a ratio that was identical to that of unliganded receptors, agonist-bound D2 receptor-ligand complexes resulted in an increase in dimerization. Addition of bivalent D2 receptor ligands also resulted in a large increase in D2 receptor dimers. A physical interaction between the protomers was confirmed using high resolution cryogenic localization microscopy, with ca. 9 nm between the centers of mass.

The influence of different cellular environments on PET radioligand binding: an application to D2/3-dopamine receptor imaging.

Various D2/3 receptor PET radioligands are sensitive to endogenous dopamine release in vivo. The Occupancy Model is generally used to interpret changes in binding observed in in vivo competition binding studies; an Internalisation Hypothesis may also contribute to these changes in signal. Extension of in vivo competition imaging to other receptor systems has been relatively unsuccessful. A greater understanding of the cellular processes underlying signal changes following endogenous neurotransmitter release may help translate this imaging paradigm to other receptor systems. To investigate the Internalisation Hypothesis we assessed the effects of different cellular environments, representative of those experienced by a receptor following agonist-induced internalisation, on the binding of three D2/3 PET ligands with previously reported sensitivities to endogenous dopamine in vivo, namely [3H]spiperone, [3H]raclopride and [3H]PhNO. Furthermore, we determined the contribution of each cellular compartment to total striatal binding for these D2/3 ligands. These studies suggest that sensitivity to endogenous dopamine release in vivo is related to a decrease in affinity in the endosomal environment compared with those found at the cell surface. In agreement with these findings we also demonstrate that ∼25% of total striatal binding for [3H]spiperone originates from sub-cellular, microsomal receptors, whereas for [3H]raclopride and [3H]PhNO, this fraction is lower, representing ∼14% and 17%, respectively. This pharmacological approach is fully translatable to other receptor systems. Assessment of affinity shifts in different cellular compartments may play a crucial role for understanding if a radioligand is sensitive to endogenous release in vivo, for not just the D2/3, but other receptor systems.

Dopaminergic receptors in the human skin.

Dopamine is a neurotransmitter which plays an important role in many human organs including the skin. In this study we will examine the presence and the distribution of D1 and D2 dopamine receptors in a particular zone of the human skin. Samples of the human plantar skin were harvested during autopsies after the consent of relatives of the dead donors. In this study the following experimental procedures were performed: 1) drawing of the human plantar skin; 2) cutting of tissues; 3) staining of tissues; 4) staining of the nerve fibres; 5) radio-binding methods for labelling D1 and D2 dopamine receptors; 6) light microscope autoradiography; 7) quantitative analysis of images and 8) statistical analysis of data. The dopamine receptors D1 are distributed particularly in the dermis layer of the human plantar skin. They are numerous in lower epidermal layers (with exclusion of the corneal layer) and few in subcutaneous tissue. On the contrary D2 dopamine receptors are prominent in the subcutaneous tissue near the vessels. Quantitative analysis of images and statistical analysis of the data confirm all our results. The specific distribution of D1 and D2 dopamine receptors in the human plantar skin is in close relation with the functions of a particular zone of the human skin that supports the weight of all the body. Moreover the character of dopamine receptors distribution is very important for further understanding the role of these receptors in the human skin.

Frightening music triggers rapid changes in brain monoamine receptors: a pilot PET study.

UNLABELLED: Frightening music can rapidly arouse emotions in listeners that mimic those from actual life-threatening experiences. However, studies of the underlying mechanism for perceiving danger created by music are limited. METHODS: We investigated monoamine receptor changes induced by frightening music using (11)C-N-methyl-spiperone ((11)C-NMSP) PET. Ten healthy male volunteers were included, and their psychophysiologic changes were evaluated. RESULTS: Compared with the baseline condition, listening to frightening music caused a significant decrease in (11)C-NMSP in the right and left caudate nuclei, right limbic region, and right paralimbic region; a particularly significant decrease in the right anterior cingulate cortex; but an increase in the right frontal occipital and left temporal lobes of the cerebral cortex. CONCLUSION: Transient fright triggers rapid changes in monoamine receptors, which decrease in the limbic and paralimbic regions but increase in the cerebral cortex.

Identification of dopamine receptor in Tetrahymena thermophila by fluorescent ligands.

Two types dopamine receptor present in the cell membrane of vertebrates. But in this study D1 receptor was identified in the invertebrate ciliates protozoan, Tetrahymena thermophila by use of fluorescent ligands. D1 specific agonist SKF-38393 binds specifically to Tetrahymena. The specific binding of SKF-38393 was encountered by equimolar addition of D1 antagonist thus showed no binding of ligands. In addition, it was also proved that the D1 specific agonist did not cross bind with the D2 type receptor due to the equimolar addition of D2 selective antagonist spiperone. Interestingly this study also showed that the dopamine receptor present in the endoplasmic reticulum and endosomes of Tetrahymena as well as cell membrane which was revealed by laser scanning microscope. Therefore, this evidence supports the existence of a D1 receptor in the ciliate protozoan.

Amphetamine regulates NR2B expression in Go2α knockout mice and thereby sustains behavioral sensitization.

The α-subunit of Go2 is a regulator of dopamine (DA) homeostasis. Deletion of the protein results in an imbalance of the direct and indirect DA pathway by reducing D1 and increasing D2 receptors. As a result, cocaine-induced behavioral sensitization is abolished. Here we show that repeated amphetamine injections in Go2α-/- mice induced a similar D1/D2 receptor ratio shift as cocaine but surprisingly the knockouts developed normal behavioral sensitization. DA receptor signaling following either cocaine or amphetamine treatment was also similar in Go2α-/- mice suggesting another mechanism involved in the differential behavioral response. Evidence is increasing that DA-glutamate interactions in the striatum determine psychostimulant action. In this line, repeated amphetamine injections led to a twofold increase in the amount of the NMDA receptor subunit NR2B in Go2α-/- mice resulting in an enhanced inhibition of the indirect DA pathway. This effect is not seen after cocaine treatment. Furthermore, amphetamine but not cocaine treatment maintained the ratio between the glutamate receptor mGluR1/5 interacting proteins Homer and Homer1a in the knockouts thereby sustaining the direct pathway. We conclude that amphetamine provokes behavioral sensitization in Go2α-/- mice by an enhanced inhibition of the indirect pathway without disturbing the direct pathway thereby overcoming the imbalance in the DArgic system.

Concentration of receptor and ligand revisited in a modified receptor binding protocol for high-affinity radioligands: [3H]Spiperone binding to D2 and D3 dopamine receptors.

In receptor binding assays with ultra-high-affinity radioligands, it is difficult, in practice, to adhere the golden rule that the receptor concentration in the assay should be substantially (at least 10-fold) lower than the dissociation constant (K(d)) of the radioligand and inhibition constant (K(i)) of compound. Especially for low specific activity radioligands (usually tritiated ligands of a couple of TBq/mmol), routinely applied in concentrations at around or below the K(d), the use of extremely small amounts of receptor protein per assay will result in low levels of bound radioactivity; the alternative use of larger assay volumes will make it difficult to apply 96-well filtration devices. For assessing the inhibition constant (K(i)) of competitive inhibitors under conditions violating the above golden rule, equations are available incorporating both [receptor] and [ligand] versus K(d); however, their application requires precise knowledge of [receptor] or initial bound/free [radioligand] ratio. In this study, we present the theoretical basis for determining the K(i) for a competitive inhibitor in a new protocol at high [protein] and high [radioligand] with the simple Cheng-Prusoff correction without the need to correct for [receptor] or initial bound/free [radioligand] ratio. In addition, we present results on the binding of the ultra-high-affinity ligand [(3)H]spiperone to dopamine D(2) and D(3) receptors validating the K(i) values calculated with the new protocol for competitive inhibitors as compared with those calculated with the most comprehensive equation available to date, that of Munson and Rodbard (1988). Binding was measured at varying [radioligand] and [receptor], test compounds (including (-)5-OH-DPAT, (+/-)7-OH-DPAT, and ropinirole) were run with varying [receptor], and simulations were done at vastly varying [radioligand] for inhibitors with vastly different K(i)s. The modified high [radioligand] protocol presented here removes a major hindrance in the proper execution of binding assays with ultra-high-affinity tritiated ligands with K(d) values in the sub-nanomolar range, allowing the use of 96-well plates with small volumes of 100-200 microl per binding assay.

MDMA-evoked changes in the binding of dopamine D(2) receptor ligands in striatum of rats with unilateral serotonin depletion.

We earlier reported an anomalous 50% decrease in [(11)C]N-methylspiperone ([(11)C]NMSP) binding to dopamine D(2)-like receptors in living pig striatum after challenge with 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"), suggesting either (1) a species peculiarity in the vulnerability of butyrophenone binding to competition from dopamine or (2) a novel consequence of synergistic actions of serotonin and dopamine at dopamine receptors. To distinguish these possibilities, we used microPET to test the vulnerability of [(11)C]NMSP binding in striatum of rats with unilateral telencephalic serotonin lesions, later verified by [(125)I]RTI-55 autoradiography. Baseline [(11)C]NMSP microPET recordings were followed by either saline or MDMA-HCl (4 mg/kg) injections (i.v.), and a second [(11)C]NMSP recording, culminating with injection of [(3)H]raclopride for autoradiography ex vivo. Neither MDMA-challenge nor serotonin lesion had any detectable effect on [(11)C]NMSP binding. In contrast, MDMA challenge increased receptor occupancy by [(3)H]raclopride ex vivo (relative to the B(max) in vitro) from 8% to 12%, and doubled the free ligand concentration in cerebral cortex, apparently by blocking hepatic CYP2D6. Assuming a single binding-site model, the increased [(3)H]raclopride binding indicated doubling of the apparent equilibrium dissociation constant in vivo (K(app) (d)), revealing a 2-fold increase in competition from endogenous dopamine at [(3)H]raclopride binding sites. The results favor hypothesis (1) that the remarkable vulnerability of [(11)C]NMSP binding in pig striatum to MDMA challenge does not generalize to the rodent.

Non-competitive interaction between raclopride and spiperone on human D-receptors in intact Chinese hamster ovary cells.

We recently investigated the binding properties of the antagonists [(3)H]-raclopride and [(3)H]-spiperone to intact Chinese hamster ovary cells expressing recombinant human D(2long)-dopamine receptors (CHO-D(2L) cells). Compared with saturation binding with [(3)H]-raclopride, raclopride reduced [(3)H]-spiperone binding with to low potency in competition binding experiments. The present findings illustrate the ability of spiperone to inhibit [(3)H]-raclopride binding non-competitively. While raclopride only decreases the apparent K(D) of [(3)H]-raclopride in saturation binding experiments, spiperone only decreases the number of sites to which [(3)H]-raclopride binds with high affinity. Also, while the IC(50) of raclopride depends on the concentration of [(3)H]-raclopride in competition experiments, this is not the case for spiperone. Kinetic studies reveal that the binding of raclopride at its high affinity sites does not affect the association of subsequently added [(3)H]-spiperone nor the rebinding of freshly dissociated [(3)H]-spiperone to the same or surrounding receptors. Yet, spiperone does not affect the dissociation rate of [(3)H]-raclopride and raclopride does not affect the (genuine) dissociation rate of [(3)H]-spiperone. The easiest way to interpret the present findings in molecular terms is to assume that D(2L)-receptors or their dimeric complexes possess two distinct binding sites: one with high affinity/accessibility for [(3)H]-raclopride and the other one with high affinity/accessibility for [(3)H]-spiperone. The ability of bound spiperone to inhibit high affinity raclopride binding while the reverse is not the case suggests for the occurrence of non-reciprocal allosteric interactions. These new findings could point at the occurrence of allosteric interactions between different classes of D(2)-receptor antagonists.

Role of the serotoninergic system in the development of diseases of the intestinal and biliferous tracts.

Primary sclerosing cholangitis and nonspecific ulcerative colitis were modeled before and after serotonin and spiperone injections. Activation of the serotoninergic system prevented the development of primary sclerosing cholangitis, but promoted more severe course of nonspecific ulcerative colitis.

Dopamine D2, D3, and D4 selective phenylpiperazines as molecular probes to explore the origins of subtype specific receptor binding.

Assembling phenylpiperazines with 7a-azaindole via different spacer elements, we developed subtype selective dopamine receptor ligands of types 1a,c, 2a, and 3a preferentially interacting with D4, D2, and D3, respectively. To complete this set, the methylthio analogues 2b and 3b exceeding the affinity of 2a and 3a by one order of magnitude and the structural intermediate 1b were synthesized. These chemically similar but biologically divergent target compounds served as molecular probes for radioligand displacement experiments, mutagenesis, and docking studies on homology models based on the recent crystal structure of the beta2-adrenergic receptor. Specific interactions with the highly conserved amino acids Asp3.32 and His6.55 and less conserved residues at positions 2.61, 2.64, 3.28, and 3.29 were identified. Inclusion of a carefully modeled extracellular loop 2 displayed two nonconserved residues in EL2 that differently contribute to ligand binding. Obviously, subtype selectivity is caused by nonconserved but frequently mediated by conserved amino acids.

Species differences in the relative densities of D1- and D2-like dopamine receptor subtypes in the Japanese quail and rats: an in vitro quantitative receptor autoradiography study.

Evidence has accumulated that the regulation of male sexual behavior by dopamine might not be the same in Japanese quail (and perhaps all birds) as it is in mammals. For example, the non-selective dopamine receptor agonist, apomorphine (APO), facilitates male sexual behavior in rats but inhibits it in quail. Although the general organization of the dopamine system is similar in birds and mammals, it is possible that the relative distribution and/or density of binding sites are different. We therefore compared the relative densities of D1-like and D2-like receptor subtypes in Japanese quail and rats, with the use of in vitro quantitative receptor autoradiography. Brain sections from 8 male rats and 8 male quail were labeled with [(3)H]SCH-23390 and [(3)H]Spiperone. In general we found a systematic species difference in the relative density of D1- vs. D2-like receptors such that the D2/D1 ratio is higher in quail than in rats in areas, known to be important target sites for dopamine action such as striatal regions or the preoptic area, which is also associated with activation of sexual behavior. This difference might explain the variation in the behavioral effectiveness of APO in rats as compared to quail; with a higher relative density of D2-like receptors in quail, a similar dose of APO would be more likely to activate inhibitory processes in quail than in rats.

Platelet dopamine: D2 receptor binding in patients with migraine.

Clinical, genetic and pharmacological evidences suggest an abnormality of the dopaminergic system in the pathogenesis of migraine. Direct evidence of an abnormal metabolism of dopamine in migraine, however, is lacking. Platelets are a useful model to understand brain dopaminergic mechanisms. The present study has been undertaken to study the status of platelet dopamine receptor binding by carrying out radioligand receptor binding assay. Binding of (3)H-spiperone to platelet membranes, known to label dopamine (DA)-D2 receptors, was conducted in 20 patients with migraine and an equal number of healthy controls. The equilibrium dissociation constant (Kd) in patients with migraine (1.71 +/- 0.19 nM) was found to be significantly lower (P < 0.001) as compared with controls (3.14 +/- 0.33 nM). However, no significant change was observed in the maximal number of binding sites (Bmax) in patients with migraine. No relationship of Kd with type of migraine, presence of vomiting, family history, frequency of attack, duration of illness and menstrual migraine was observed. The findings of the present study provide support for the involvement of the dopaminergic system in migraine.

Altered dopamine D2-like receptor binding in rats with behavioral sensitization to quinpirole: effects of pre-treatment with Ro 41-1049.

Repeated treatment with the dopamine D2/D3 receptor agonist quinpirole produces a sensitized behavioral response in rats manifested as an increase in locomotor activity. Pre-treatment with certain monoamine oxidase inhibitors, such as Ro 41-1049 [N-(2-aminomethyl)-5-(3-fluorophenyl)-4-thiazolecarboxamide HCl], changes the sensitized response from locomotion to stationary, self-directed mouthing. In this study, the effects of quinpirole sensitization, with and without pre-treatment with Ro 41-1049, were determined on dopamine D2-like receptors in the nucleus accumbens and the striatum. Long-Evans rats were pre-treated with Ro 41-1049 (1 mg/kg) 90 min prior to administration of quinpirole (0.5 mg/kg, 8 injections, every 3-4 days). Dopamine D2-like receptor binding was determined 3 days after the last injection by ex vivo radioligand assays using [3H]spiperone and [3H]quinpirole. Densities of [3H]spiperone- and [3H]quinpirole-labeled sites were both increased 32% in the nucleus accumbens of rats with demonstrated locomotor sensitization to quinpirole. In contrast, the density of dopamine D2-like receptors in quinpirole-sensitized rats pre-treated with Ro 41-1049 was not different from saline controls. These findings support the involvement of alterations in dopamine D2-like receptors in the development of locomotor sensitization to quinpirole and suggest that modification of these alterations in dopamine D2-like receptors contributes to the change from sensitized locomotion to mouthing observed when rats are pre-treated with Ro 41-1049.

Mechanisms for metoclopramide-mediated sensitization and haloperidol-induced catalepsy in rats.

Typical antipsychotics such as the dopamine D(2) receptor antagonist, haloperidol are known to cause movement disorders or catalepsy in experimental animals. Catalepsy is believed to result from blockade of dopamine D(2) receptors. In this study two drugs that differ in antipsychotic potency but are similar in blocking dopamine D(2) receptors were used to investigate the mechanism for catalepsy and its sensitization. Metoclopramide is a strong postsynaptic dopamine D(2) receptor blocker with no antipsychotic potency. At low doses of 5 or 10 mg/kg given subcutaneously (s.c.), metoclopramide did not produce catalepsy or movement disturbance for seven days after drug treatment. Also metoclopramide at 10 mg/kg given for five days, failed to induce catalepsy. Haloperidol, another potent dopamine D(2) receptor blocker at 0.5 mg/kg (s.c.) rapidly produced catalepsy and suppressed movement 1 h after a single dose of the drug. Chronic as well as acute treatment with metoclopramide caused sensitization of haloperidol-induced catalepsy. Neurochemical analyses revealed significant dopamine D(2) receptor up-regulation in both frontal cortex and striatum of rats chronically treated with metoclopramide. However, no changes in dopamine D(2) receptor numbers were noted in these areas after chronic treatment with low doses of haloperidol. Significant increases in N-methyl-D-aspartate (NMDA) receptor numbers were observed in both frontal cortex and striatum of metoclopramide treated animals, while haloperidol elicited significant decreases in NMDA receptor numbers in both brain areas. These observations plus previous reports have led us to propose a model for catalepsy and its sensitization. According to this model the increase in NMDA receptors by metoclopramide sensitizes the brain to haloperidol-induced catalepsy. Thus, catalepsy appears to be elicited by simultaneous activation of glutamatergic NMDA and dopamine D(1) receptors as well as a blockade of dopamine D(2) receptors.

Antagonist-radioligand binding to D2L-receptors in intact cells.

D(2)-dopamine receptors mediate most of the physiological actions of dopamine and are important recognition sites for antipsychotic drugs. Earlier binding studies were predominantly done with broken cell preparations with the tritiated D(2)-receptor antagonists [(3)H]-raclopride, a hydrophilic benzamide, and [(3)H]-spiperone, a highly hydrophobic butyrophenone. Here we compared [(3)H]-raclopride and [(3)H]-spiperone binding properties in intact Chinese Hamster Ovary cells stably expressing recombinant human D(2L)-receptors. Specific binding of both radioligands occurred to a comparable number of sites. In contrast to the rapid dissociation of [(3)H]-raclopride in both medium only and in the presence of an excess of unlabelled ligand [(3)H]-spiperone dissociation was only observed in the latter condition, and it was still slower than in broken cell preparations. However, this could not explain the pronounced difference in the potency of some unlabelled ligands to compete with both radioligands. To integrate these new findings, a model is proposed in which raclopride approaches the receptor from the aqueous phase, while spiperone approaches the receptor by lateral diffusion within the membrane.

Dopamine D2(High) receptors on intact cells.

In humans, behavioral dopamine supersensitivity occurs in schizophrenia and in Parkinson's disease. In animals, behavioral dopamine supersensitivity is consistently associated with increased dopamine D2(High) receptors in homogenized striata in vitro. Because D2(High) receptors have not yet been detected in intact cells, we used [(3)H]domperidone to detect D2(High) sites in intact rat anterior pituitary adenoma culture cells. Although [(3)H]raclopride and [(3)H]spiperone did not detect D2(High) receptors in intact cells or in rat fresh striatal slices, [(3)H]domperidone readily detected D2(High) receptors, warranting an in vivo search for D2(High) variations in human diseases.

PET analysis of the 5-HT2A receptor inverse agonist ACP-103 in human brain.

The mechanisms underlying the clinical properties of atypical antipsychotics have been postulated to be mediated, in part, by interactions with the 5-HT2A receptor. Recently, it has been recognized that clinically effective antipsychotic drugs are 5-HT2A receptor inverse agonists rather than neutral antagonists. In the present study, which is part of the clinical development of the novel, selective 5-HT2A receptor inverse agonist ACP-103, we applied positron emission tomography (PET) with the radioligand [11C]N-methylspiperone ([11C]NMSP) to study the relationship between oral dose, plasma level, and uptake of ACP-103 in living human brain. The safety of drug administration was also assessed. Four healthy volunteers were examined by PET at baseline, and after the oral administration of various single doses of ACP-103. Two subjects each received 1, 5, and 20 mg doses, and two subjects each received 2, 10, and 100 mg doses, respectively. ACP-103 was well tolerated. Detectable receptor binding was observed at very low ACP-103 serum levels. Cortical [11C]NMSP binding was found to be dose-dependent and fitted well to the law of mass action. A reduction in binding was detectable after an oral dose of ACP-103 as low as 1 mg, and reached near maximal displacement following the 10-20 mg dose. In conclusion, administration of ACP-103 to healthy volunteers was found to be safe and well tolerated, and single oral doses as low as 10 mg were found to fully saturate 5-HT2A receptors in human brain as determined by PET.