Isolation and quantitative analysis of B<Subscript>1</Subscript>, B<Subscript>2</Subscript>, B<Subscript>6</Subscript> AND B<Subscript>12</Subscript> vitamins using high-performance thin-layer chromatography

A new, simple and accurate high-performance thin-layer chromatography (HPTLC) method has been developed for the separation and simultaneous analysis of B1, B2, B6 and B12 vitamins. Standard and sample solutions of vitamins were applied onto precoated aluminum-backed silica gel G 60 F254 HPTLC plate. The plates were developed with about thirty different solvent systems, and the ethanol – chloroform – acetonitrile – toluene – ammonia – water (7 : 4 : 4.5 : 0.5 : 1 : 1, v/v) mixture was chosen as the optimum mobile phase. UV detection was performed densitometrically at 254 nm. The retention factors of B1, B2, B6 and B12 vitamins were 0.36, 0.60, 0.85 and 0.46, respectively. The linearity range for indicated compounds was 10 – 2500 ng per spot and the correlation coefficients were R ² = 0.97 – 0.98. The limits of quantification (LOQ) were 141.72, 42.41, 100.31 and 11.50 ng and the limits of detection (LOD) were 42.52, 12.72, 30.09 and 3.45 ng for B1, B2, B6 and B12 vitamins, respectively. Analyses of standard solutions showed good precision and repeatability.     

Interactions of recombinant human histamine H<Subscript>1</Subscript>, H<Subscript>2</Subscript>, H<Subscript>3</Subscript>, and H<Subscript>4</Subscript> receptors with 34 antidepressants and antipsychotics

Antidepressants and antipsychotics affect multiple molecular targets. Consequently, these drugs exhibit not only unique profiles of therapeutic effects but also several undesired effects. Histamine receptors (H₁R, H₂R, H₃R, and H₄R) belong to the large family of G protein-coupled receptors and are very important drug targets. All four H _x R subtypes are expressed in the CNS. Interactions of lipophilic, blood–brain barrier-penetrating drugs with H _x Rs could contribute to therapeutic and unwanted effects. Therefore, we investigated potencies H _x R as well as potencies and (inverse) agonistic efficacies of 34 antidepressants and antipsychotics at HxRs in functional assays. We expressed human H _x Rs in Sf9 insect cells and conducted radioligand competition binding experiments and functional steady-state GTPase assays. Ligand affinities and potencies were compared with literature data and related to therapeutic reference ranges. Almost all antidepressants and antipsychotics displayed high binding affinities to H₁R and behaved as antagonists. The atypical antidepressant trimipramine behaved as a high-affinity/high-potency H₂R antagonist (pK _i, 7.39; pK _B, 7.36; pA ₂, 7.55). Docking to an H₂R model suggested a probable binding mode. The affinity of antidepressants and antipsychotics for H₃R was low. The atypical antipsychotic clozapine, known to induce agranulocytosis, exhibited partial H₄R agonism for which docking experiments provided a molecular basis. Clozapine also exhibited H₂R antagonism. We observed dissociations between pK _i and pK _B values as well as between pK _i and pIC₅₀ values for H _x Rs. Antidepressants and antipsychotics interact differentially with H _x Rs. The concept of functional selectivity (also referred to as ligand-specific receptor conformations or biased signaling) explains dissociations between pK _i and pK _B values as well as differences between pK _i and pIC₅₀ values. The H₁R antagonism of numerous antidepressants and antipsychotics is very pronounced. The H₂R antagonism of trimipramine and partial H₄R agonism of clozapine may be clinically relevant. We also discuss the possible role of the H₂R antagonism of clozapine for neutropenia/agranulocytosis induced by this compound. Finally, we discuss the methodological, conceptual, and clinical limitations of our study.     

Cytotoxic purine nucleoside analogues bind to A<Subscript>1</Subscript>, A<Subscript>2A</Subscript>, and A<Subscript>3</Subscript> adenosine receptors

Fludarabine, clofarabine, and cladribine are anticancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A₁, A_2A, A_2B, and A₃). Radioligand binding studies utilizing Chinese hamster ovary cells, stably transfected with adenosine A₁, A_2A, or A₃ receptor subtype, were used to assess the binding affinities of these compounds, whereas adenylyl cyclase activity was used to assess the binding to A_2B receptors. Clofarabine and cladribine both bound to the A_2A receptor with a K _i of 17 and 15 μM, respectively. Clofarabine was the only adenosine analogue to bind to the A₃ receptor with a K _i of 10 μM, and none of these compounds bound to the A_2B receptor. Results show that clofarabine, cladribine, and fludarabine bind to the A₁ receptor. In addition, clofarabine, cladribine, and fludarabine were A₁ agonists (IC₅₀ 3.1, 30, and 30 μM, respectively). Neither pyrimidine nucleoside analogues gemcitabine nor cytarabine associated with any of the adenosine receptor subtypes (K _i > 100μM). This is the first report of an interaction between all adenosine receptor subtypes and chemotherapeutic nucleoside analogues commonly used in the treatment of cancer. Therefore, activation of these receptors may be at least one mechanism through which fludarabine-associated toxicity occurs.     

Pharmacological characterization of mitogen-activated protein kinase activation by recombinant human 5-HT<Subscript>2C</Subscript>, 5-HT<Subscript>2A</Subscript>, and 5-HT<Subscript>2B</Subscript> receptors

The type 2 serotonin (5-HT₂) receptor subfamily is known to couple to phosphoinositide hydrolysis (PI) and the subsequent mobilization of intracellular Ca²⁺, as well as the release of arachidonic acid (AA). Less is known of 5-HT₂-mediated activation of the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK1/2) signaling. The present study measured the relative efficacies and potencies of 5-HT agonists to activate ERK2 in non-neuronal cells expressing recombinant human 5-HT_2A, 5-HT_2B, and 5-HT_2C(ISV) receptors. 5-HT agonists stimulated ERK2 activity via all three 5-HT₂ subtypes. There were no meaningful differences in the potencies or relative efficacies of these agonists to affect ERK2 activity vs. PI accumulation or Ca²⁺ mobilization, suggesting that these pathways may be sequentially linked. Indeed, ERK2 activity was very sensitive to PKC inhibition and calcium chelation and insensitive to tyrosine kinase and PI-3-kinase inhibition. 5-HT₂ receptors efficiently couple to MAPK activation via sequential PI hydrolysis, and Ca²⁺ mobilization. This profile differs from reports of “agonist-directed trafficking of receptor stimulus” between PI/Ca²⁺ and AA pathways activated by 5-HT₂ receptors.     

Pharmacological characterisation of the agonist radioligand binding site of 5-HT<Subscript>2A</Subscript>, 5-HT<Subscript>2B</Subscript> and 5-HT<Subscript>2C</Subscript> receptors

In the present study we compared the affinity of various drugs for the high affinity agonist-preferring binding site of human recombinant 5-HT_2A, 5-HT_2B and 5-HT_2C receptors stably expressed in monoclonal mammalian cell lines. To ensure that the agonist-preferring conformation of the receptor was preferentially labelled in competition binding experiments, saturation analysis was conducted using antagonist and agonist radiolabels at each receptor. Antagonist radiolabels ([³H]-ketanserin for 5-HT_2A receptor and [³H]-mesulergine for 5-HT_2B and 5-HT_2C receptor) bound to a larger population of receptors in each preparation than the corresponding agonist radiolabel ([¹²⁵I]-DOI for 5-HT_2A receptor binding and [³H]-5-HT for 5-HT_2B and 5-HT_2C receptor binding). Competition experiments were subsequently conducted against appropriate concentrations of the agonist radiolabels bound to the agonist-preferring subset of receptors in each preparation. These studies confirmed that there are a number of highly selective antagonists available to investigate 5-HT₂ receptor subtype function (for example, MDL 100907, RS-127445 and RS-102221 for 5-HT_2A, 5-HT_2B and 5-HT_2C receptors respectively). There remains, however, a lack of highly selective agonists. (–)DOI is potent and moderately selective for 5-HT_2A receptors, BW723C86 has poor selectivity for human 5-HT_2B receptors, while Org 37684 and VER-3323 display some selectivity for the 5-HT_2C receptor. We report for the first time in a single study, the selectivity of numerous serotonergic drugs for 5-HT₂ receptors from the same species, in mammalian cell lines and using, exclusively, agonist radiolabels. The results indicate the importance of defining the selectivity of pharmacological tools, which may have been over-estimated in the past, and highlights the need to find more selective agonists to investigate 5-HT₂ receptor pharmacology.     

Non-CB<Subscript>1</Subscript>, Non-CB<Subscript>2</Subscript> Receptors for Endocannabinoids,Plant Cannabinoids,and Synthetic Cannabimimetics: Focus on G-protein-coupled Receptors and Transient Receptor Potential Channels

The molecular mechanism of action of Δ⁹-tetrahydrocannabinol (THC), the psychotropic constituent of Cannabis, has been a puzzle during the three decades separating its characterization, in 1964, and the cloning, in the 1990s, of cannabinoid CB₁ and CB₂ receptors. However, while these latter proteins do mediate most of the pharmacological actions of THC, they do not seem to act as receptors for other plant cannabinoids (phytocannabinoids), nor are they the unique targets of the endogenous lipids that were originally identified in animals as agonists of CB₁ and CB₂ receptors, and named endocannabinoids. Over the last decade, several potential alternative receptors for phytocannabinoids, endocannabinoids, and even synthetic cannabimimetics, have been proposed, often based uniquely on pharmacological evidence obtained in vitro. In particular, the endocannabinoid anandamide, and the other most abundant Cannabis constituent, cannabidiol, seem to be the most “promiscuous” of these compounds. In this article, we review the latest data on the non-CB₁, non-CB₂ receptors suggested so far for endocannabinoids and plant or synthetic cannabinoids, and lay special emphasis on uncharacterized or orphan G-protein-coupled receptors as well as on transient receptor potential channels.     

Occupancy of dopamine D<Subscript>1</Subscript>, D<Subscript>2</Subscript> and serotonin<Subscript>2A</Subscript> receptors in schizophrenic patients treated with flupentixol in comparison with risperidone and haloperidol

Rationale Flupentixol (FLX) has been used as a neuroleptic for nearly 4 decades. In vitro data show comparable affinity to dopamine D₂, D₁ and 5-HT_2A receptors and recently, FLX showed to be not inferior to risperidone in schizophrenic patients with predominant negative symptomatology, which was implicated with flupentixol’s interaction with 5-HT_2A and/or D₁ receptors. Objectives To assess in vivo receptor occupancy (RO) in patients clinically treated with FLX (n = 13, 5.7 ± 1.4 mg/day) in comparison with risperidone (RIS, n = 11, 3.6 ± 1.3 mg/day) and haloperidol (HAL, n = 11, 8.5 ± 5.5 mg/day). Materials and methods Each patient underwent two PET scans with 3-N-[¹¹C]methylspiperone (target: frontal 5-HT_2A), [¹¹C]SCH23390 (striatal D₁) or [¹¹C]raclopride (striatal D₂). RO was calculated as the percentage reduction of specific binding in comparison with healthy controls. Results D₂-RO under FLX was between 50% and 70%, indicating an ED₅₀ of about 0.7 ng/ml serum. 5-HT_2A and D₁-RO was 20 ± 10% and 20 ± 5% (mean, SEM). Under HAL, D₁-RO was 14 ± 6% and under RIS not significantly different from zero. Conclusions We were able to demonstrate a moderate 5-HT_2A and D₁ occupancy under clinically relevant doses of flupentixol, albeit lower than expected from in vitro data and clearly below saturation. Therefore, if flupentixol’s efficacy on negative symptoms is based on its interaction with 5-HT_2A and/or D₁ receptors, it should be highly dependent on serum concentration and thus on dosage and metabolism. However, these data suggest that mechanisms other than D₁ or 5-HT_2A antagonism may contribute to flupentixol’s efficacy on negative symptoms.     

Synthesis of Allylthiopyridazine Derivatives and Inhibition of Aflatoxin B<Subscript>1</Subscript>-Induced Hepatotoxicity in Rats

Five kinds of allylthiopyridazine derivatives were synthesized and their chemoprotective activities examined in rats exposed to aflatoxin B1-toxicant. Rats were pretreated with five allylthiopyridazine derivatives at daily oral doses of 50 mg/kg for 10 consecutive days, and during this period with one or three repeated doses of the potent hepatotoxin, aflatoxin B1. The hepatoprotective effects of the allylthiopyridazine derivatives against aflatoxin B1 (1 mg/kg, three times at intervals of 3 days, i.p., or at 3 mg/kg, once at final days, i.p.) administration were showed the significantly normal as compared with control in body and liver weights. Aspartate aminotransferase and alanine aminotransferase levels were markedly elevated after aflatoxin B1 administration, and pretreatment with allylthiopyridazine derivatives, before aflatoxin B1 administration, resulted in decreased levels of these enzymes. In addition, the allylthiopyridazine derivatives, K6 (3-methoxy-), K8 (3-chloro-), K16 (3-ethoxy-) and K17 (3-n-propoxy), induced elevated hepatic GSH levels. Four kinds of allylthiopyridazine derivatives investigated were effective against aflatoxin B1-induced hepatotoxicity.     

Rewarding effects elicited by cocaine microinjections into the ventral tegmental area of C57BL/6 mice: involvement of dopamine D<Subscript>1</Subscript> and serotonin<Subscript>1B</Subscript> receptors

Rationale The role of ventral tegmental area (VTA) in mediating the rewarding effects of cocaine has not been extensively studied.Objectives We used the intracranial self-administration (ICSA) procedure to assess the involvement of the VTA in the rewarding effects of cocaine, and the effect of dopamine (DA) D₁- and serotonin (5-HT)_1B-receptor antagonists on ICSA of cocaine.Methods Adult male C57BL/6 mice were stereotaxically implanted, unilaterally, with a guide cannula either 1.5 or 2.3 mm above the VTA. After 1 week, mice were trained to discriminate between the two arms of a Y-maze over seven daily sessions, one arm being reinforced by intracranial cocaine microinjections. Starting from session 8, the D₁ and 5-HT_1B-receptor antagonists were injected IP pre-test each day over five consecutive sessions.Results Mice injected into the VTA rapidly exhibited a preference for the cocaine-reinforced arm, whatever the dose of cocaine available (30 pmol or 150 pmol per injection), reaching optimum ICSA performance within 5 days. In contrast, mice injected 0.8 mm above the VTA did not discriminate between the arms of the maze and performed at random, except for one subject. Once the ICSA response was acquired, systemic pre-injections of either the D₁ (SCH23390; 25 g/kg IP) or 5-HT_1B (GR127935; 0.5 mg/kg IP) antagonist disrupted this behavior. Replacement of each antagonist by vehicle led to the reinstatement of intra-VTA cocaine self-administration.Conclusions The results of the present study suggest that VTA neurons play a critical role in mediating the rewarding effects of acute cocaine and that both D₁ and 5-HT_1B receptors modulate these effects.     

Cytotoxic and genotoxic effects of fumonisin B<Subscript>1</Subscript> on rabbit kidney RK13 cell line

Fumonisins, mycotoxins produced by certain strains of Fusarium moniliforme, could induce various diseases in animals and are suspected human carcinogens. Fumonisin B1 (FB1), the most commonly found fumonisin, has been characterised as a tumour initiator and a tumour promoter, a mitogen and an anti-proliferative agent. In this study we examined the cytotoxicity and genotoxicity of FB1 in rabbit kidney RK13 cells. To evaluate the effects of FB1 on survival of this cell line we analysed cell viability, membrane integrity, DNA fragmentation and overall morphology of the cells. The genotoxic potential of FB1 was estimated by monitoring the ability of this mycotoxin to induce micronuclei in RK13 cells. Exposure to FB1 caused a significant increase in micronucleus frequency in a concentration- and in a time-dependent manner. Nanomolar concentrations of FB1 decreased cell viability after 24 h and even more so after 48 h of exposure. The morphological changes observed suggested that an increased number of RK13 cells were dying by the process of apoptosis. However, FB1 also induced impairments of cell and mitochondrial membrane integrity, as assessed by lactate dehydrogenase and glutamate dehydrogenase leakage. These results could imply that nanomolar concentrations of FB1 induced apoptosis, which subsequently may proceed to secondary necrosis. In summary, our observations suggest that FB1 is genotoxic and cytotoxic to RK13 cells.     

Further characterization of the 5-HT<Subscript>1</Subscript> receptors mediating cardiac sympatho-inhibition in pithed rats: pharmacological correlation with the 5-HT<Subscript>1B</Subscript> and 5-HT<Subscript>1D</Subscript> subtypes

Serotonin (5-hydroxytryptamine; 5-HT) is capable of inhibiting the tachycardic responses elicited by sympathetic stimulation, but not by exogenous noradrenaline, in pithed rats pre-treated with desipramine. More recently, it has been shown that this cardiac sympatho-inhibitory response to 5-HT, mediated by prejunctional 5-HT₁ receptors as well as putative 5-ht_5A/5B receptors, is mimicked dose-dependently by the agonists CP 93,129 (r5-HT_1B), sumatriptan (5-HT_1B/1D) and PNU-142633 (5-HT_1D). This study analysed further the pharmacological profile of the above 5-HT₁ receptors.Continuous i.v. infusions of CP 93,129, sumatriptan or PNU-142633 (30 µg kg^–1min^–1 each) failed to modify the tachycardic responses to exogenous noradrenaline but inhibited those elicited by preganglionic (C7–T1) stimulation of the cardiac sympathetic outflow. These sympatho-inhibitory responses were unaltered after i.v. administration of physiological saline (1 ml kg^–1) or the 5-HT_1A receptor antagonist WAY 100635 (10 µg kg^–1). In contrast, the antagonist GR 127935 (5-HT_1B/1D; 100 µg kg^–1, i.v.) abolished the responses to CP 93,129, sumatriptan and PNU-142633, whilst SB224289 (5-HT_1B; 300 µg kg^–1, i.v.) abolished the responses to CP 93,129 without affecting those to sumatriptan and PNU-142633. Interestingly, BRL15572 (5-HT_1D; 300 µg kg^–1, i.v.) abolished the responses to PNU-142633 and attenuated those to sumatriptan, but not those to CP 93,129.WAY 100635, GR 127935, SB224289 and BRL15572, given alone at the above doses, failed to modify the sympathetically induced tachycardic responses. The 5-HT₁ receptors producing cardiac sympatho-inhibition in pithed rats thus display the pharmacological profile of the 5-HT_1B and 5-HT_1D receptor subtypes.     

DNA damage in fetal liver cells of turkey and chicken eggs dosed with aflatoxin B<Subscript>1</Subscript>

The present study was undertaken to investigate the potential of the potent hepatocarcinogen aflatoxin B₁ (AFB) to produce DNA damage in turkey and chicken fetal livers in ovo. Effects of a single injection of two different doses (0.062 and 6.2 μg) of AFB were examined under both short-term (4 h) and longer-term (4 day) dosing of eggs from turkeys at 24 days and chickens at 18 days of development. Liver cells prepared from the fetuses were used to assess the extent of DNA damage by the alkaline single-cell gel electrophoresis (comet) assay. The results demonstrate that AFB produces dose-related DNA damage in the fetal livers of both turkeys and chicken at 4 h, which was reduced by 4 days. Turkey embryos appeared to be slightly more susceptible to AFB damage, although no difference in the survival between chicken and turkey fetuses was observed.     

<Emphasis Type="Italic">NAT2</Emphasis>,<Emphasis Type="Italic"> CYP2C9</Emphasis>,<Emphasis Type="Italic"> CYP2C19</Emphasis>, and<Emphasis Type="Italic"> CYP2E1</Emphasis> genetic polymorphisms in anti-TB drug-induced maculopapular eruption

Purpose  

It has been suggested that drug-metabolizing enzymes might play important roles in the development of anti-tuberculosis drug (ATD)-induced maculopapular eruption (MPE), as in ATD-induced hepatitis. We investigated the associations between the genetic polymorphisms of drug-metabolizing enzymes and ATD-induced MPE.     

Evaluation of Bioadhesive Capacity and Immunoadjuvant Properties of Vitamin B<Subscript>12</Subscript>-Gantrez Nanoparticles

Purpose  To design bioadhesive Gantrez AN (poly[methyl vinyl ether-co-maleic anhydride], PVM/MA) nanoparticles (NP) coated with vitamin B₁₂ (Vit B₁₂), and investigate their application in oral antigen delivery. Methods  The association of Vit B₁₂ to Gantrez AN nanoparticles was performed by the direct attachment of reactive Vit B₁₂ to the surface of the nanoparticles (NPB), or linking to the copolymer chains in dimethylformamide prior to NP formation (NPB-DMF). Nanoparticles were characterized by measuring the size, zeta potential, Vit B₁₂ association efficacy, and stability of Vit B₁₂ on the surface of the nanoparticles. In vivo bioadhesion study was performed by the oral administration of fluorescently-labeled nanoparticle formulations to rats. Both systemic and mucosal immune responses were evaluated after oral and subcutaneous immunization with ovalbumin (OVA) containing Vit B₁₂-coated nanoparticles. Results  The Vit B₁₂ nanoparticles displayed homogenous size distribution with a mean diameter of about 200 nm and a negative surface charge. The association efficiency of Vit B₁₂ to NPB-DMF formulation was about two times higher than to the NPB, showing also a higher surface stability of Vit B₁₂. The bioadhesion study demonstrated that NPB-DMF had an important tropism to the distal portions of the gut, which was about two and 3.5 times higher than the tropism observed for NPB and control NP, respectively (p < 0.05). Oral administration of OVA-NPB-DMF induced also stronger and more balanced serum anti-OVA titers of IgG2a (Th1) and IgG1 (Th2) compared to control OVA-NP. In addition, oral immunization with OVA-NPB-DMF induced a higher mucosal IgA response than subcutaneous administration. Conclusions  These results indicate the benefits of bioadhesive Vit B₁₂-coated nanoparticles in oral antigen delivery eliciting systemic and mucosal immune response.     

Immunodensity and mRNA expression of A<Subscript>2A</Subscript> adenosine,D<Subscript>2</Subscript> dopamine,and CB<Subscript>1</Subscript> cannabinoid receptors in postmortem frontal cortex of subjects with schizophrenia: effect of antipsychotic treatment

Rationale  

Dopamine D₂ receptors are the main target of antipsychotic drugs. In the brain, D₂ receptors coexpress with adenosine A_2A and CB₁ cannabinoid receptors, leading to functional interactions.     

Protective effects of three luteolin derivatives on aflatoxin B<Subscript>1</Subscript>-induced genotoxicity on human blood cells

In the present study, we aimed to investigate the genotoxic and anti-genotoxic potencies of three luteolin derivatives (luteolin-7-O-glucoside, luteolin-7-O-rutinoside and luteolin-7-O-glucuronide) by using human cells. In the micronucleus test, the human lymphocytes were exposed to aflatoxin B₁, the luteolin derivatives and a mixture of the two for 72?h. Furthermore, we have evaluated the levels of antioxidants of human whole blood plasma in order to clarify the possible mechanisms that may contribute to the anti-genotoxic activity of the luteolin derivatives. According to the results obtained from the micronucleus test, the highest protection rates for luteolin-7-O-glucoside, luteolin-7-O-rutinoside and luteolin-7-O-glucuronide against aflatoxin B1 were 32.09, 35.55 and 37.50?%, respectively. Similarly, these three luteolin derivatives ameliorated the level of antioxidants altered from aflatoxin B₁.     

Ischemic and pharmacological preconditioning induces further delayed protection in transgenic mouse cardiac myocytes over-expressing adenosine A<Subscript>1</Subscript> receptors (A<Subscript>1</Subscript>AR): role of A<Subscript>1</Subscript>AR,iNOS and K<Subscript>ATP</Subscript> channels

In this study we examined the hypotheses that over-expression of the adenosine A(1) receptor (A(1)AR) in transgenic mouse cardiac myocytes (A(1)AR-tgm) induces cellular protection against subsequent sustained simulated ischemia (SI); that the cellular protection induced by over-expression of A(1)AR in A(1)AR-tgm is mediated through inducible nitric oxide synthase (iNOS) and K(ATP) channels. Sub-lethal simulated ischemia (SSI) and the A(1)AR agonists chloro- N(6)-cyclopentyl adenosine (CCPA) or (2 S)- N(6)-[2-endo-norbornyl]adenosine (S-ENBA) induce further, delayed cytoprotection, additional to the existing protection in A(1)AR-tgm. Cellular injury and cell viability was measured by the release of lactate dehydrogenase (LDH) or creatine kinase (CK) into the medium and the amount remaining in the cells. The cellular resistance acquired by cardiac myocytes due to the over-expression of A(1)AR was reflected by the reduced release of LDH (in units/liter) from 44.94+/-1.46 (wild-type mouse cardiac myocytes, wt) to 29.59+/-2.83 (A(1)AR-tgm, P<0.001). Conversely, LDH release from A(1)AR-tgm increased to 42.53+/-2.23 ( P<0.01) on exposure to 5-hydroxydecanoate (a mitochondrial K(ATP) channel blocker), to 45.93+/-2.90 ( P<0.01) on exposure to S-methylthiourea (an iNOS inhibitor) and to 56.04+/-3.00 ( P<0.01) on exposure to glibenclamide (a K(ATP) channel blocker). Treatment of A(1)AR-tgm is with SSI and the A(1)AR agonists chloro- N(6)-cyclopentyl adenosine (CCPA) or (2 S)- N(6)-[2-endo-norbornyl]adenosine (S-ENBA) decreased the release of LDH from 46.44+/-0.57 (A(1)AR-tgm) to 42.08+/-0.48 (A(1)AR-tgm plus SSI, P<0.05), 38.03+/-1.16 (A(1)AR-tgm plus CCPA, P<0.001) and 32.77+/-0.58 (A(1)AR-tgm plus S-ENBA, P<0.001). Our data suggest that the A(1)AR has a cytoprotective effect against subsequent sustained SI in A(1)AR-tgm and that the cellular protection induced by over-expression of A(1)AR in A(1)AR-tgm depends on iNOS and K(ATP) channels. Further, SSI and the A(1)AR agonists CCPA or S-ENBA induce further, delayed cytoprotection in A(1)AR-tgm.     

Differential regulation of rat peripheral 5-HT<Subscript>2A</Subscript> and 5-HT<Subscript>2B</Subscript> receptor systems: influence of drug treatment

Most studies of 5-HT₂ receptor regulation have been carried out on the central nervous system (CNS) (which expresses 5-HT_2A and 5-HT_2C receptors); very few in vitro studies have addressed the peripheral receptors 5-HT_2A and 5-HT_2B. The aim of this investigation was to compare the possible short- and long-term processes regulating these peripheral receptors in the rat.The in vitro contractile response elicited by serotonin (5-HT, 10 µM) in the rat gastric fundus (5-HT_2B receptor system) was rapid and followed by a partial fade to a steady state, in contrast with the rat thoracic aorta response (5-HT_2A receptor system), which was more stable, slower and sustained. To characterize drug-receptor interactions, cumulative concentration/response curves (CCRCs) for 5-HT were constructed ex vivo for rat tissues treated with drugs acting at these receptors. Rats were examined 4 or 24 h after a single, i.p. administration of (±)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(±)DOI, 1 or 2.5 mg/kg], clozapine, cyproheptadine or rauwolscine (10 mg/kg), 48 h after a single i.p. administration of (±)DOI (2.5 mg/kg), clozapine or cyproheptadine (10 mg/kg) or 24 h after the last of with 15 daily i.p. administrations of (±)DOI (1 or 2.5 mg/kg), clozapine, cyproheptadine or rauwolscine (10 mg/kg). In the aorta, E_max (the maximum response elicited by 5-HT) was unchanged 4 h after a single dose of any of the drugs tested. However, 24 h after a single dose, E_max was lower in animals treated with (±)DOI (2.5 mg/kg), clozapine or cyproheptadine than in controls, whilst 48 h after a single dose of (±)DOI (2.5 mg/kg), clozapine or cyproheptadine there was no difference in E_max between experimental and control animals. After chronic treatment with (±)DOI (2.5 mg/kg), clozapine and cyproheptadine, E_max was lower than in controls. In the gastric fundus, E_max 4 h after a single dose of each drug was lower than in controls, and the response recovered by 24 or 48 h. Following chronic treatment, E_max was significantly lower than in controls for each drug used.These findings suggest first, that regulation of peripheral 5-HT₂ receptors (5-HT_2A and 5-HT_2B) is a functionally significant phenomenon in vivo, and occurs after administration of both agonists and antagonists. Second, the kinetics of peripheral 5-HT₂ receptor regulation were similar in both in vivo and ex vivo experiments. The 5-HT_2B receptors in rat gastric fundus are more sensitive to drug-induced regulation than the 5-HT_2A rat aortic receptors. Finally, long-term regulation of both receptors stabilizes short-term desensitization for longer.     

Non-uniform blockade of intrastriatal D<Subscript>2</Subscript>/D<Subscript>3</Subscript> receptors by risperidone and amisulpride

Rationale Atypical antipsychotic drugs have been shown to preferentially affect extrastriatal (mesolimbic) D₂/D₃ receptors over those within the striatum (nigrostriatal). The striatum does not contain exclusively nigrostriatal dopamine tracts, however. The caudate nucleus and ventral parts of the striatum primarily contain limbic and associative dopamine pathways more relevant to psychosis. Objectives We tested the hypothesis that two pharmacologically distinct atypical antipsychotic drugs, amisulpride and risperidone, would preferentially occupy of D₂/D₃ dopamine receptors in limbic and associative regions of the striatum. Methods Eight amisulpride-treated patients, six risperidone-treated patients and six age- and sex-matched healthy controls were recruited. Dynamic SPET studies were performed after bolus injection of [¹²³I]epidepride. Binding potential (BP) images were generated using a modified Logan method and aligned between subjects. Regions of interest (ROIs) were placed around head of caudate and putamen bilaterally on an average BP map derived from aligned control images. These ROIs were then applied user-independently to the BP maps for each subject to calculate BP for head of caudate and putamen. Mean occupancy of D₂/D₃ receptors in each ROI was determined by reference to the drug-free healthy volunteer group. Occupancy values for head of caudate and putamen were compared using paired Student’s t test. Results D₂/D₃ receptor occupancy was 42% in caudate and 31% in putamen for risperidone (t=5.9, df=11, p=0.0001) and 51% in caudate and 37% in putamen for amisulpride (t=11.1, df=15, p<0.0001). Conclusions Amisulpride and risperidone both show selective occupancy for limbic and associative D₂/D₃ receptors within the striatum.     

Dual and selective antagonism of neurokinin NK<Subscript>1</Subscript> and NK<Subscript>2</Subscript> receptor-mediated responses in human colon mucosa

The neurokinin (NK) receptors, NK(1) and NK(2), which are activated by substance P (SP) and NKA, have been identified as potential therapeutic targets in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Here we have investigated the effects of a novel dual NK(1) and NK(2) receptor antagonist, namely DNK333 upon responses elicited by [Sar(9), Met(O(2))(11)]-SP (SMSP) and [betaAla(8)]-NKA(4-10) in isolated human colon mucosa mounted in Ussing chambers. A selective NK(1) receptor antagonist, SR140333 and NK(2) receptor antagonist, SR48968 have been tested for comparison. Additions of SMSP (100 nM) or [betaAla(8)]-NKA(4-10) (100 nM) increased basal short-circuit current and responses to both peptides were inhibited by DNK333, while SR140333 only inhibited SMSP and SR48968 blocked only [betaAla(8)]-NKA(4-10) responses. SR140333 did not attenuate [betaAla(8)]-NKA(4-10) effects and SR48968 had no effect upon SMSP responses. Carbachol (1 micro M) responses were not altered by any of the three NK antagonists. We conclude that activation of either NK(1) or NK(2) receptors can stimulate epithelial ion transport in human colon mucosa and that the novel dual antagonist, DNK333 may be of potential therapeutic interest in the treatment of IBD and IBS.