Showing papers in "British Journal of Pharmacology in 1999"

Suppression of nitric oxide synthase and the down-regulation of the activation of NFκB in macrophages by resveratrol

Abstract: Resveratrol, naringenin and naringin are naturally occurring flavonoids in grapes and grapefruits. The anti-inflammatory effects of these flavonoids have been well documented, but the mechanism is poorly characterized. High concentration of NO are produced by inducible NO synthase (iNOS) in inflammation, and the prevention of the expression of iNOS may be an important anti-inflammatory mechanism. In this study, the effects of these flavonoids on the induction of NO synthase (NOS) in RAW 264.7 cells activated with bacterial lipopolysaccharide (LPS, 50 ng ml(-1)) were investigated. Resveratrol was found strongly to inhibit NO generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic iNOS protein and steady state mRNA levels. However, the inhibitory abilities of naringenin were lower, and the inhibitory abilities of naringin were almost negligible. In electrophoretic mobility shift assays, the activation of NFkappaB induced by LPS for 1 h was inhibited by resveratrol (30 microM). Furthermore, in immunoblotting analysis, cells treated with LPS plus resveratrol showed an inhibition of phosphorylation as well as degradation of IkappaBalpha, and a reduced nuclear content of NFkappaB subunits. The flavonoids may be of value for inhibiting the enhanced expression of iNOS in inflammation through down-regulation of NFkappaB binding activity.

Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656, and AM630.

Abstract: We have tested our prediction that AM630 is a CB2 cannabinoid receptor ligand and also investigated whether L759633 and L759656, are CB2 receptor agonists. Binding assays with membranes from CHO cells stably transfected with human CB1 or CB2 receptors using [3H]-CP55940, confirmed the CB2-selectivity of L759633 and L759656 (CB2/CB1 affinity ratios = 163 and 414 respectively) and showed AM630 to have a Ki at CB2 receptors of 31.2 nM and a CB2/CB1 affinity ratio of 165. In CB2-transfected cells, L759633 and L759656 were potent inhibitors of forskolin-stimulated cyclic AMP production, with EC50 values of 8.1 and 3.1 nM respectively and CB1/CB2 EC50 ratios of > 1000 and > 3000 respectively. AM630 inhibited [35S]-GTPgammaS binding to CB2 receptor membranes (EC50 = 76.6 nM), enhanced forskolin-stimulated cyclic AMP production in CB2-transfected cells (5.2 fold by 1 microM), and antagonized the inhibition of forskolin-stimulated cyclic AMP production in this cell line induced by CP55940. In CB1-transfected cells, forskolin-stimulated cyclic AMP production was significantly inhibited by AM630 (22.6% at 1 microM and 45.9% at 10 microM) and by L759633 at 10 microM (48%) but not 1 microM. L759656 (10 microM) was not inhibitory. AM630 also produced a slight decrease in the mean inhibitory effect of CP55940 on cyclic AMP production which was not statistically significant. We conclude that AM630 is a CB2-selective ligand that behaves as an inverse agonist at CB2 receptors and as a weak partial agonist at CB1 receptors. L759633 and L759656 are both potent CB2-selective agonists.

Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells

Abstract: 1. The goal of this study was to characterize the agonist pharmacology of human 5-HT2A, 5-HT2B and 5-HT2C (VSV) receptors expressed in CHO-K1 (Chinese hamster ovary) cells. 2. We used a fluorometric imaging plate reader (FLIPR) which allows rapid detection of rises in intracellular calcium levels upon the addition of agonists. 3. Stimulation of all three receptors by 5-HT caused a robust concentration dependent increase in intracellular calcium levels. No such effect was observed from non-transfected control CHO-K1 cells. 4. The rank order of potency of agonists at the different receptor subtypes varied. Tryptamines, BW-723C86, d-norfenfluramine, Ro 60-0175 and LSD exhibited the following rank order of potency; 5-HT2B>5-HT2C>5-HT2A. Piperazines such as m-Chlorophenylpiperazine (mCPP), ORG-12962, MK-212 and also ORG-37684 exhibited a rank order of potency of 5-HT2C>5-HT2B>5-HT2A. The phenylisopropylamines DOI and DOB had a rank order of 5-HT2A>5-HT2B>5-HT2C. 5. Many agonists tested had partial agonist actions when compared to 5-HT, and a wide range of relative efficacies were exhibited, which was cell line dependent. For example, mCPP had a relative efficacy of 65% at 5-HT2C receptors but <25% at either 5-HT2A or 5-HT2B receptors. 6. Interpretation of literature values of functional assays using different cell lines, different receptor expression levels and different receptor isoforms, is complex. Species differences and the previous use of antagonist radioligands to characterize agonist potency in binding assays emphasizes the importance of studying agonists in the same experiment using the same assay conditions and parental cell lines.

Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy

Abstract: Cyclo-oxygenase is expressed in cells in two distinct isoforms. Cyclo-oxygenase-1 is present constitutively whilst cyclo-oxygenase-2 is expressed primarily after inflammatory insult. The activity of cyclo-oxygenase-1 and -2 results in the production of a variety of potent biological mediators (the prostaglandins) that regulate homeostatic and disease processes. Inhibitors of cyclo-oxygenase include the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin, ibuprofen and diclofenac. NSAIDs inhibit cyclo-oxygenase-2 at the site of inflammation, to produce their therapeutic benefits, as well as cyclo-oxygenase-1 in the gastric mucosa, which produces gastric damage. Most recently selective inhibitors of cyclo-oxygenase-2 have been developed and introduced to man for the treatment of arthritis. Moreover, recent epidemiological evidence suggests that cyclo-oxygenase inhibitors may have important therapeutic relevance in the prevention of some cancers or even Alzheimer's disease. This review will discuss how the new advancements in NSAIDs research has led to the development of a new class of NSAIDs that has far reaching implications for the treatment of disease.

Distribution and regulation of cyclooxygenase-2 in carrageenan-induced inflammation.

Abstract: 1 We characterized the regulation of cyclooxygenase-2 (COX-2) at the mRNA, protein and mediator level in two rat models of acute inflammation, carrageenan-induced paw oedema and mechanical hyperalgesia. 2 Carrageenan was injected in the hind paw of rat at low (paw oedema) and high doses (hyperalgesia). COX-2 and prostaglandin E2 (PGE2) levels were measured by RT-PCR and immunological assays. We also determined the distribution of COX-2 by immunohistochemistry. 3 The injection of carrageenan produced a significant and parallel induction of both COX-2 and PGE2. This induction was significantly higher in hyperalgesia than in paw oedema. This was probably due to the 9 fold higher concentration of carrageenan used to provoke hyperalgesia. 4 Immunohistochemical examination showed COX-2 immunoreactivity in the epidermis, skeletal muscle and inflammatory cells of rats experiencing hyperalgesia. In paw oedema however, only the epidermis showed positive COX-2 immunoreactivity. 5 Pretreatment with indomethacin completely abolished the induction of COX-2 in paw oedema but not in hyperalgesia. 6 These results suggest that multiple mechanisms regulate COX-2 induction especially in the more severe model. In carrageenan-induced paw oedema, prostanoid production have been linked through the expression of the COX-2 gene which suggest the presence of a positive feedback loop mechanism.

Leukocyte-endothelial cell adhesion: avenues for therapeutic intervention

Abstract: Leukocyte-endothelial cell adhesion has been implicated in the pathogenesis of a variety of diseases that affect different organ systems. Examples of such diseases include atherosclerosis, gastric ulcers, haemorrhagic shock, myocardial infarction, stroke, and malaria (Korthuis et al., 1994; Panes & Granger, 1998). The recognition that leukocytes must firmly adhere to vascular endothelial cells in order to mediate the organ dysfunction and tissue injury associated with these diseases has resulted in an intensive effort to define the factors that modulate this cell-cell interaction. A major focal point of this effort has been directed towards identifying and characterizing the adhesion glycoproteins that enable leukocytes to bind to vascular endothelial cells. Data derived from both in vitro (isolated leukocytes binding to monolayers of cultured endothelial cells) and in vivo (intravital microscopic examination of venules) models of leukocyte-endothelial cell adhesion have revealed the relative contributions of different leukocyte and endothelial cell adhesion molecules (CAMs) to the adhesion responses elicited by various inflammatory stimuli. These studies have also led to an appreciation of the potential cellular and molecular loci that can be targeted to interfere with leukocyte-endothelial cell adhesion. As a result, there is a widely held view that several of these loci also represent novel and potentially powerful therapeutic sites for treatment of acute and chronic inflammatory diseases. The major objective of this review is to discuss some of the major potential targets for therapeutic intervention against inflammation that relate to the process of leukocyte-endothelial cell adhesion and more specifically to the regulation of endothelial CAM expression. That discussion is preceded by a brief description of the major CAMs that participate in the recruitment of leukocytes into inflamed tissue and how the expression of these CAMs is coordinated to ensure an orderly sequence of cell-cell interactions.

Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis-associated properties in A431 cells overexpressing epidermal growth factor receptor

Abstract: 1. Flavonoids display a wide range of pharmacological properties including anti-inflammatory. Anti-mutagenic, anti-carcinogenic and anti-cancer effects. Here, we evaluated the effects of eight flavonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion. 2. Of the flavonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and significantly inhibited A431 cell proliferation with IC50 values of 19 and 21 micronM, respectively. 3. The epidermal growth factor (EGF) (10 nM) promoted growth of A431 cells (+25+/-4.6%) and mediated epidermal growth factor receptor (EGFR) tyrosine kinase activity and autophosphorylation of EGFR were inhibited by Lu and Qu. At concentration of 20 micronM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR. 4. A431 cells treated with Lu or Qu exhibited protuberant cytoplasmic blebs and progressive shrinkage morphology. Lu and Qu also time-dependently induced the appearance of a ladder pattern of DNA fragmentation, and this effect was abolished by EGF treatment. 5. The addition of EGF only marginally diminished the inhibitory effect of luteolin and quercetin on the growth rate of A431 cells, treatment of cellular proteins with EGF and luteolin or quercetin greatly reduced protein phosphorylation, indicating Lu and Qu may act effectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase. 6. EGF increased the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), while Lu and Qu appeared to suppress the secretion of these two MMPs in A431 cells. 7. Examination of the relationship between the chemical structure and inhibitory effects of eight flavonoids reveal that the double bond between C2 and C3 in ring C and the OH groups on C3' and C4' in ring B are critical for the biological activities. 8. This study demonstrates that the inhibitory effects of Lu and Qu, and the stimulatory effects of EGF, on tumour cell proliferation, cellular protein phosphorylation, and MMP secretion may be mediated at least partly through EGFR. This study supports the idea that Lu and Qu may have potential as anti-cancer and anti-metastasis agents.

ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1.

Abstract: The present study was performed to investigate the ability of the multidrug resistance protein (MRPI) to transport different cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1-transfected cell line (S1(MRP)). As substrates we used 3H-labelled derivatives of the hydrophilic monoquaternary cation N-(4',4'-azo-in-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. ATP- and GSH-dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1-specific monoclonal antibody QCRL-3. MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations > or = 10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 microM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP- and GSH-dependent manner. APDA is not a substrate for MRP1.

The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein

Abstract: 1 The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [3H]-XR9576 with the multidrug resistance P-glycoprotein (P-gp). XR9576 displayed specific high-affinity binding to P-gp (Bmax = 275 pmol mg-1, Kd = 5.1 nM). The transport substrates [3H]-vinblastine and [3H]-paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P-gp. The duration of action of XR9576 with P-gp was increased in comparison to that of vinblastine which displayed a slower rate of association and a faster dissociation rate. 2 The relative affinities of several modulators and transport substrates to interact with P-gp were determined from displacement drug equilibrium binding assays. Vinblastine and paclitaxel could only fractionally displace [3H]-XR9576 binding, displaying Ki values significantly different from their measured Kd values. This suggests a non-competitive interaction between XR9576 and the P-gp substrates vinblastine and paclitaxel. 3 XR9576 was shown to be a potent modulator of P-gp mediated [3H]-vinblastine and [3H]-paclitaxel transport as it increased the steady-state accumulation of these cytotoxics in CHrB30 cells to levels observed in non-P-gp-expressing AuxB1 cells (EC50 = 487+/-50 nM). This inhibition of drug transport is not mediated through competition for transport since [3H]-XR9576 accumulation was not influenced by P-gp expression or function. 4 These results demonstrate that the P-gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this transporter than any other reported modulators. Several lines of evidence suggest that XR9576 inhibits P-gp function by binding at a site which is distinct from the site of interaction of transport substrates. The two sites may be classified as serving modulatory or transport functions.

Pharmacological characterization of the human P2Y11 receptor

Abstract: 1 The human P2Y11 receptor is coupled to both the phosphoinositide and the cyclic AMP pathways A pharmacological characterization of the recombinant human P2Y11 receptor has been conducted following stable expression in two different cell lines: the 1321N1 astrocytoma cells for inositol trisphosphate measurements and the CHO-K1 cells for cyclic AMP assays The rank order of potency of a series of nucleotides was almost identical for the two pathways: ATPgammaS approximately BzATP > dATP > ATP > ADPbetaS > 2MeSATP 2 ADPbetaS, AMPalphaS and A3P5PS behaved as partial agonists of the human P2Y11 receptor At high concentrations, these three nucleotides were able to partially inhibit the ATP response 3 Suramin was a more potent antagonist than reactive blue 2, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid was completely inactive The P2Y11 receptor proved to be sensitive to suramin in a competitive way with an apparent Ki value of 082+/-0 07 microM 4 The ATP derivative AR-C67085 (2-propylthio-beta, gamma-dichloromethylene-D-ATP), a potent inhibitor of ADP-induced platelet aggregation, was the most potent agonist of the P2Y11 receptor, among the various nucleotides tested 5 The pharmacological profile of the recombinant human P2Y11 receptor is closely similar to that of the cyclic AMP-coupled P2 receptor recently described in HL-60 cells, suggesting that it is the same receptor

The recombinant 5-HT1A receptor: G protein coupling and signalling pathways.

Abstract: The 5-hydroxytryptamine 5-HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5-HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5-HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5-HT1A receptor in the decade since the identificiation of its cDNA.

Inhibition by simvastatin, but not pravastatin, of glucose-induced cytosolic Ca2+ signalling and insulin secretion due to blockade of L-type Ca2+ channels in rat islet β-cells

Abstract: 1. Hypercholesterolaemia often occurs in patients with type 2 diabetes, who therefore encounter administration of HMG-CoA reductase inhibitors. Alteration of pancreatic beta-cell function leading to an impaired insulin secretory response to glucose plays a crucial role in the pathogenesis of type 2 diabetes. Therefore, it is important to examine the effects of HMG-CoA reductase inhibitors on beta-cell function. 2. Cytosolic Ca2+ concentration ([Ca2+]i) plays a central role in the regulation of beta-cell function. The present study examined the effects of HMG-CoA reductase inhibitors on the glucose-induced [Ca2+]i signalling and insulin secretion in rat islet beta-cells. 3. Simvastatin, a lipophilic HMG-CoA reductase inhibitor, at 0.1-3 microg ml(-1) concentration-dependently inhibited the first phase increase and oscillation of [Ca2+]i induced by 8.3 mM glucose in single beta-cells. The less lipophilic inhibitor, simvastatin-acid, inhibited the first phase [Ca2+]i increase but was two orders of magnitude less potent. The hydrophilic inhibitor, pravastatin (100 microg ml(-1), was without effect on [Ca2+]i. 4. Simvastatin (0.3 microg ml(-1)), more potently than simvastatin-acid (30 microg ml(-1)), inhibited glucose-induced insulin secretion from islets, whereas pravastatin (100 microg ml(-1)) had no effect. 5. Whole-cell patch clamp recordings demonstrated a reversible inhibition of the beta-cell L-type Ca2+ channels by simvastatin, but not by pravastatin. Simvastatin also inhibited the [Ca2+]i increases by L-arginine and KCl, agents that act via opening of L-type Ca2+ channels. 6. In conclusion, lipophilic HMG-CoA reductase inhibitors can inhibit glucose-induced [Ca2+]i signalling and insulin secretion by blocking L-type Ca2+ channels in beta-cells, and their inhibitory potencies parallel their lipophilicities. Precaution should be paid to these findings when HMG-CoA reductase inhibitors are used clinically, particularly in patients with type 2 diabetes.

Functional and molecular biological evidence for a possible β3‐adrenoceptor in the human detrusor muscle

Abstract: The possible existence of a beta3-adrenergic receptor (beta3-AR) in the human detrusor muscle was investigated by in vitro functional studies and analysis of mRNA expression. Isoprenaline, noradrenaline and adrenaline each produced a concentration-dependent relaxation of the human detrusor. The rank order for their relaxing potencies was isoprenaline (pD2 6.37+/-0.07) > or = noradrenaline (pD2 6.07+/-0.12) > or = adrenaline (pD2 5.88 M) and its pKB value was 5.71+/-0.19. Moreover, SR58894A (10(-7) - 10(-5) M), a beta3-AR antagonist, caused a rightward shift of the concentration-relaxation curve for isoprenaline in a concentration-dependent manner. The pA2 value and slope obtained from Schild plots were 6.24+/-0.20 and 0.68+/-0.31. The beta1-, beta2- and beta3-AR mRNAs were all positively expressed in detrusor smooth muscle preparations in a reverse transcription polymerase chain reaction assay. In conclusion, the present results provide the first evidence for the existence of the beta3-AR subtype in the human detrusor. They also suggest that the relaxation induced by adrenergic stimulation of the human detrusor is mediated mainly through beta3-AR activation.

Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR2)-activating peptides in the rat paw.

Abstract: In the present study, we have observed the development of an inflammatory reaction in the rat hindpaw, following the injection of specific agonists of PAR2 (two PAR2 activating peptides). This inflammation was characterized by oedema and granulocyte infiltration. Two selective PAR2 activating peptides, SLGRL-NH2 and trans-cinnamoyl-LIGRLO-NH2 induced significant oedema in the rat hindpaw from 1-6 h following subplantar injection. Six hours after the PAR2-activating peptide injection, the paw tissues showed a complete disruption of tissue architecture along with an inflammatory cell infiltrate. In the inflamed paw, PAR2-immunoreactivity was expressed on endothelial cells as well as on the infiltrating inflammatory cells. The oedema induced by the injection of the two PAR2 activating peptides was slightly reduced in rats pre-treated with compound 48/80, but was not modified by pre-treatment of rats with cromolyn, a mast cell stabilizer. Pre-treatment of rats with a cyclo-oxygenase inhibitor (indomethacin) or a nitric oxide synthase inhibitor (L-N(omega)-nitro-L-arginine methyl ester) had no effect on the oedema induced by the PAR2-activating peptides. These results demonstrate that the administration of PAR2-activating peptides into the rat paw induced an acute inflammatory response characterized by a persistent oedema (at least 6 h) and granulocyte infiltration. The PAR2-induced inflammatory response occurred through a mechanism largely independent of mast cell activation, and of the production of prostanoids and nitric oxide.

Role of the medial prefrontal cortex in 5-HT1A receptor-induced inhibition of 5-HT neuronal activity in the rat.

Abstract: We examined the involvement of the frontal cortex in the 5-HT1A receptor-induced inhibition of 5-HT neurones in the dorsal raphe nucleus (DRN) of the anaesthetized rat using single-unit recordings complemented by Fos-immunocytochemistry. Both transection of the frontal cortex as well as ablation of the medial region of the prefrontal cortex (mPFC) significantly attenuated the inhibition of 5-HT neurones induced by systemic administration of the 5-HT1A receptor agonist, 8-OH-DPAT (0.5–16 μg kg−1, i.v.). In comparison, the response to 8-OH-DPAT was not altered by ablation of the parietal cortex. The inhibitory effect of 8-OH-DPAT was reversed by the 5-HT1A receptor antagonist, WAY 100635 (0.1 mg kg−1, i.v.) in all neurones tested. In contrast, cortical transection did not alter the sensitivity of 5-HT neurones to iontophoretic application of 8-OH-DPAT into the DRN. Similarly, cortical transection did not alter the sensitivity of 5-HT neurones to systemic administration of the selective 5-HT reuptake inhibitor, paroxetine (0.1–0.8 mg kg−1, i.v.). 8-OH-DPAT evoked excitation of mPFC neurones at doses (0.5–32 μg kg−1, i.v.) in the range of those which inhibited 5-HT cell firing. At higher doses (32–512 μg kg−1, i.v.) 8-OH-DPAT inhibited mPFC neurones. 8-OH-DPAT (0.1 mg kg−1, s.c.) also induced Fos expression in the mPFC. The neuronal excitation and inhibition, as well as the Fos expression, were antagonized by WAY 100635. These data add further support to the view that the inhibitory effect of 5-HT1A receptor agonists on the firing activity of DRN 5-HT neurones involves, in part, activation of a 5-HT1A receptor-mediated postsynaptic feedback loop centred on the mPFC. Keywords: Serotonin, 5-HT1A receptor, 8-OH-DPAT, WAY 100635, paroxetine, medial prefrontal cortex, dorsal raphe nucleus Introduction It is well established that exogenous 5-HT or 5-HT1A receptor agonists inhibit the activity of midbrain 5-HT neurones via activation of somatodendritic 5-HT1A receptors. Thus, local iontophoretic application of 5-HT or 5-HT1A receptor agonists like 8-OH-DPAT lead to cessation of spontaneous firing of 5-HT neurones recorded in the dorsal raphe nucleus (DRN) of both anaesthetized and awake animals (Aghajanian et al., 1972; Sprouse & Aghajanian, 1987; Levine & Jacobs, 1992). Furthermore, these treatments have the same effect when applied directly onto 5-HT neurones recorded in brain slice or dissociated cell preparations (Aghajanian & Lakoski, 1984; Penington et al., 1993a,1993b). The inhibitory effect of selective 5-HT re-uptake inhibitor (SSRI) antidepressants on 5-HT neurones has also been attributed to the activation of somatodendritic 5-HT1A autoreceptors (Blier & de Montigny, 1983; Gartside et al., 1995; Hajos et al., 1995a), and it is presumed that this inhibition contributes to the lack of antidepressant effect early on in the treatment (Blier & de Montigny, 1994). Interestingly, there is accumulating evidence that 5-HT1A receptor agonists might inhibit the activity of 5-HT neurones in the DRN not only via somatodendritic 5-HT1A autoreceptors but also 5-HT1A receptors located at other sites. Earlier studies reported that chronic treatment of rats with lithium (Blier et al., 1987), the 5-HT1A receptor agonists gepirone or ipsapirone (Blier & de Montigny, 1987; Schechter et al., 1990), or SSRIs (Blier & de Montigny, 1980; Jolas et al., 1994), attenuates the inhibitory effect on 5-HT neurones of iontophoretically administered 5-HT or 8-OH-DPAT. Similar results were obtained when 5-HT1A receptor agonists are applied to brain slices prepared from antidepressant treated animals (Jolas et al., 1994). In contrast, antidepressant treatment does not modify the inhibitory effect of 8-OH-DPAT when the drug is administered systemically (Blier & de Montigny, 1987; Schechter et al., 1990; Jolas et al., 1994). These findings suggest that (i) 5-HT1A receptors other than those located on the somatodendrites of 5-HT neurones might regulate 5-HT neuronal activity and (ii) in contrast to somatodendritic 5-HT1A receptors, these 5-HT1A receptors (presumably located postsynaptically in relation to 5-HT nerve terminals) do not desensitize upon chronic antidepressant treatment. A recent paper reported more direct evidence for a role of postsynaptic 5-HT1A receptors in the inhibition of 5-HT neurones by systemically administered 8-OH-DPAT. Thus, Ceci et al. (1994) reported that after an acute, deep transection of the frontal cortex and forebrain, the dose-response curve for the inhibitory effect of 8-OH-DPAT on the firing 5-HT neurones was shifted 10 fold to the right. Although this study did not resolve the anatomical location of the postsynaptic 5-HT1A receptors, the dense population of 5-HT1A receptors located in the medial prefrontal cortex (mPFC; Pompeiano et al., 1992; Kia et al, 1996) was a likely candidate. Interestingly, we have recently demonstrated that there is a direct projection from the mPFC to the DRN, and that electrical stimulation of this pathway inhibited most DRN 5-HT neurones tested (Hajos et al., 1998b). Here we further investigate the role of the frontal cortex, and the mPFC in particular, in the 8-OH-DPAT-induced inhibition of 5-HT neuronal activity. Experiments include the use of animals with acute cortical/mPFC lesions to test of the effects of 8-OH-DPAT and the SSRI, paroxetine administered systemically or iontophoretically on DRN 5-HT neurones. The effects of 8-OH-DPAT on neuronal activity in the mPFC was measured electrophysiologically and by Fos protein immunocytochemistry. In most experiments the effect of 8-OH-DPAT was validated pharmacologically using the selective 5-HT1A receptor antagonist, WAY 100635 (Fletcher et al., 1996). A preliminary account of the findings was recently presented to the Forum of European Neuroscience (Hajos et al., 1998a).

Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil

Abstract: 1. Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-beta-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using beta-glucosidase, beta-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). 2. Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. 3. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. 4. Beta-Glucosidase hydrolysed M-1 completely. Beta-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. 5. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. 6. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system.

Acetylcholine‐induced relaxation in blood vessels from endothelial nitric oxide synthase knockout mice

Abstract: 1. Isometric tension was recorded in isolated rings of aorta, carotid, coronary and mesenteric arteries taken from endothelial nitric oxide synthase knockout mice (eNOS(-/-) mice) and the corresponding wild-type strain (eNOS(+/+) mice). The membrane potential of smooth muscle cells was measured in coronary arteries with intracellular microelectrodes. 2. In the isolated aorta, carotid and coronary arteries from the eNOS(+/+) mice, acetylcholine induced an endothelium-dependent relaxation which was inhibited by N(omega)-L-nitro-arginine. In contrast, in the mesenteric arteries, the inhibition of the cholinergic relaxation required the combination of N(omega)-L-nitro-arginine and indomethacin. 3. The isolated aorta, carotid and coronary arteries from the eNOS(-/-) mice did not relax in response to acetylcholine. However, acetylcholine produced an indomethacin-sensitive relaxation in the mesenteric artery from eNOS(-/-) mice. 4. The resting membrane potential of smooth muscle cells from isolated coronary arteries was significantly less negative in the eNOS(-/-) mice (-64.8 +/- 1.8 mV, n = 20 and -58.4 +/- 1.9 mV, n = 17, for eNOS(+/+) and eNOS(-/-) mice, respectively). In both strains, acetylcholine, bradykinin and substance P did not induce endothelium-dependent hyperpolarizations whereas cromakalim consistently produced hyperpolarizations (- 7.9 +/- 1.1 mV, n = 8 and -13.8 +/- 2.6 mV, n = 4, for eNOS(+/+) and eNOS(-/-) mice, respectively). 5. These findings demonstrate that in the blood vessels studied: (1) in the eNOS(+/+) mice, the endothelium-dependent relaxations to acetylcholine involve either NO or the combination of NO plus a product of cyclo-oxygenase but not EDHF; (2) in the eNOS(-/-) mice, NO-dependent responses and EDHF-like responses were not observed. In the mesenteric arteries acetylcholine releases a cyclo-oxygenase derivative.

Oxidative stress as a mechanism for quinolinic acid-induced hippocampal damage: protection by melatonin and deprenyl.

Abstract: 1. There are differences between the excitotoxic actions of quinolinic acid and N-methyl-D-aspartate (NMDA) which suggest that quinolinic acid may act by mechanisms additional to the activation of NMDA receptors. The present study was designed to examine the effect of a potent antioxidant, melatonin, and the potential neuroprotectant, deprenyl, as inhibitors of quinolinic acid-induced brain damage. Injections were made into the hippocampus of anaesthetized rats, which were allowed to recover before the brains were taken for histology and the counting of surviving neurones. 2. Quinolinic acid (120 nmols) induced damage to the pyramidal cell layer, which was prevented by the co-administration of melatonin (5 nmols locally plus 2x20 mg kg(-1) i.p.). This protective effect was not prevented by the melatonin receptor blocker luzindole. Neuronal damage produced by NMDA (120 nmols) was not prevented by melatonin. 3. Quinolinic acid increased the formation of lipid peroxidation products from hippocampal tissue and this effect was prevented by melatonin. 4. Deprenyl also prevented quinolinic acid-induced damage at a dose of 50 nmols but not 10 nmols plus 2x1.0 mg kg(-1) i.p. The non-selective monoamine oxidase inhibitor nialamide (10 and 50 nmols plus 2x25 mg kg(-1)) did not afford protection. 5. The results suggest that quinolinic acid-induced neuronal damage can be prevented by a receptor-independent action of melatonin and deprenyl, agents which can act as a potent free radical scavenger and can increase the activity of endogenous antioxidant enzymes respectively. This suggests that free radical formation contributes significantly to quinolinic acid-induced damage in vivo.

Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L‐type Ca2+ current in rat ventricular myocytes

Abstract: The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I(Ca)) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. IBMX (100 microM), a broad-spectrum PDE inhibitor, increased basal I(Ca) by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 microM). However, MIMX (1 microM), a PDE1 inhibitor, EHNA (10 microM), a PDE2 inhibitor, cilostamide (0.1 microM), a PDE3 inhibitor, or Ro20-1724 (0.1 microM), a PDE4 inhibitor, had no effect on basal I(Ca) and little stimulatory effects on [cAMP]i (20-30%). Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I(Ca) or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40-50%), only cilostamide (0.1 microM)+ Ro20-1724 (0.1 microM) produced a significant stimulation of I(Ca) (50%). Addition of EHNA (10 microM) to this mix increased I(Ca) to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 microM) or isoprenaline (1 microM). When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I(Ca) by (approximately 40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in I(Ca). Maximal effects on I(Ca) were approximately 8% for MIMX (3 microM), approximately 20% for EHNA (1-3 microM), approximately 30% for cilostamide (0.3-1 microM) and approximately 50% for Ro20-1724 (0.1 microM). Our results demonstrate that PDE1-4 subtypes regulate I(Ca) in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I(Ca), all four PDE subtypes determine the response of I(Ca) to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3>PDE2>PDE1.

The role of P-glycoprotein in blood-brain barrier transport of morphine: transcortical microdialysis studies in mdr1a (-/-) and mdr1a (+/+) mice.

Abstract: The aim of this study was to investigate whether blood-brain barrier transport of morphine was affected by the absence of mdr1a-encoded P-glycoprotein (Pgp), by comparing mdr1a (−/−) mice with mdr1a (+/+) mice. Mdr1a (−/−) and (+/+) mice received a constant infusion of morphine for 1, 2 or 4 h (9 nmol/min/mouse). Microdialysis was used to estimate morphine unbound concentrations in brain extracellular fluid during the 4 h infusion. Two methods of estimating in vivo recovery were used: retrodialysis with nalorphine as a calibrator, and the dynamic-no-net-flux method. Retrodialysis loss of morphine and nalorphine was similar in vivo. Unbound brain extracellular fluid concentration ratios of (−/−)/(+/+) were 2.7 for retrodialysis and 3.6 for the dynamic-no-net-flux at 4 h, with corresponding total brain concentration ratios of (−/−)/(+/+) being 2.3 for retrodialysis and 2.6 for the dynamic-no-net-flux. The total concentration ratios of brain/plasma were 1.1 and 0.5 for mdr1a (−/−) and (+/+) mice, respectively. No significant differences in the pharmacokinetics of the metabolite morphine-3-glucoronide were observed between (−/−) and (+/+) mice. In conclusion, comparison between mdr1a (−/−) and (+/+) mice indicates that Pgp participates in regulating the amount of morphine transport across the blood-brain barrier. British Journal of Pharmacology (1999) 128, 563–568; doi:10.1038/sj.bjp.0702804

Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell‐line using FLIPR

Abstract: The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03±0.08 and 7.30±0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18±0.10 and 8.43±0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6–8 s), followed by a gradually declining secondary phase. Thapsigargin (3 μM) or U73122 (3 μM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx. British Journal of Pharmacology (1999) 128, 1–3; doi:10.1038/sj.bjp.0702780

Cell type‐specific ATP‐activated responses in rat dorsal root ganglion neurons

Abstract: 1. The aim of our study is to clarify the relationship between expression pattern of P2X receptors and the cell type of male adult rat (Wistar) dorsal root ganglion (DRG) neurons. We identified the nociceptive cells of acutely dissociated DRG neurons from adult rats type using capsaicin sensitivity. 2. Two types of ATP-activated currents, one with fast, the other with slow desensitization, were found under voltage-clamp conditions. In addition, cells with fast but not slow desensitization responded to capsaicin, indicating that there was a relationship between current kinetics and capsaicin-sensitivity. 3. Both types of neurons were responsive to ATP and alpha, beta methylene-ATP (alpha,betameATP). The concentration of alpha,(beta)meATP producing half-maximal activation (EC50) of neurons with fast desensitization was less (11 microM) than that of neurons with slow desensitization (63 microM), while the Hill coefficients were similar. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (PPADS) antagonized alpha,betameATP-induced currents in both types of neurons. 4. In situ hybridization revealed that small cells of the DRG predominantly expressed mRNAs of P2X3 and medium-sized cells expressed mRNAs of P2X2 and P2X3. In contrast, both of mRNAs were not detected in large cells of the DRG. 5. These results suggest that capsaicin-sensitive, small-sized DRG neurons expressed mainly the homomeric P2X3 subunit and that capsaicin-insensitive, medium-sized DRG neurons expressed the heteromultimeric receptor with P2X2 and P2X3.

Selective nitrergic neurodegeneration in diabetes mellitus–a nitric oxide‐dependent phenomenon

Abstract: In vitro and in vivo studies have demonstrated a dysfunctional nitrergic system in diabetes mellitus, thus explaining the origin of diabetic impotence. However, the mechanism of this nitrergic defect is not understood. In the penises of streptozotocin (STZ)-induced diabetic rats, here, we show by immunohistochemistry that nitrergic nerves undergo selective degeneration since the noradrenergic nerves which have an anti-erectile function in the penis remained intact. Nitrergic relaxation responses in vitro and erectile responses to cavernous nerve stimulation in vivo were attenuated in these animals, whereas noradrenergic responses were enhanced. Activity and protein amount of neuronal nitric oxide synthase (nNOS) were also reduced in the penile tissue of diabetic rats. We, thus, hypothesized that NO in the nitrergic nerves may be involved in the nitrergic nerve damage, since only the nerves which contain neuronal NO synthase underwent degeneration. We administered an inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), in the drinking water of rats for up to 12 weeks following the establishment of diabetes with STZ. Here we demonstrate that this compound protected the nitrergic nerves from morphological and functional impairment. Our results show that selective nitrergic degeneration in diabetes is NO-dependent and suggest that inhibition of NO synthase is neuroprotective in this condition. Keywords: Nitric oxide, nitrergic, diabetes mellitus, neurodegeneration, penile erection, impotence, erectile dysfunction, streptozotocin Introduction Penile erection comprises increased arterial inflow and restricted venous outflow from the penis, co-ordinated by relaxation of the penile corpus cavernosum (Lue & Tanagho, 1987). Relaxation of the cavernosal smooth muscle is regulated by local factors released from nerve fibres and the sinusoidal endothelium (Andersson & Wagner, 1995). The identity of the major mediator responsible for the relaxation of the corpus cavernosum has long been a subject for debate. Nitric oxide (NO) is now accepted as the most important relaxant mediator in the process of erection (Burnett et al., 1992; Andersson & Wagner, 1995) and other putative mediators like VIP, ATP or acetylcholine have been excluded (Wagner & Brindley, 1980; Andersson, 1993; Andersson & Wagner, 1995) In the penile corpus cavernosum NO is produced in the endothelium and nitrergic nerves (Moncada et al., 1997) by endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) respectively. Although acetylcholine evokes endothelium-dependent relaxation of the isolated corpus cavernosum (Saenz de Tejada et al., 1988), erection is unlikely to involve release of acetylcholine from parasympathetic nerves and its subsequent diffusion to the endothelium to release NO, since neither atropine nor neostigmine inhibit erection when injected intracavernosally (Wagner & Brindley, 1980; Brindley, 1986; Burnett et al., 1992). Furthermore, relaxation of the corpus cavernosum by transmural stimulation does not require functional endothelium (Saenz de Tejada et al., 1988; Kim et al., 1991; Okamura et al., 1999). Thus the majority of NO eliciting relaxation in penile tissue seems to be derived from nNOS in nitrergic nerves. In vivo and in vitro studies suggest that the nitrergic system is essential for penile erection (Andersson & Wagner, 1995). In anaesthetized animals, erection induced by stimulation of the cavernous nerve or spinal cord can be inhibited by NOS inhibitors (Holmquist et al., 1991; Burnett et al., 1992; Trigo-Rocha et al., 1993; Finberg et al., 1993; Andersson & Wagner, 1995). Corpus cavernosum obtained from patients relaxes to transmural stimulation in vitro, an effect that can be blocked by NOS inhibitors (Bush et al., 1992; Holmquist et al., 1992). Erectile dysfunction is caused by many diseases, one of which is diabetes mellitus; the prevalence of impotence in diabetic patients is up to 50% (Rubin & Babbott, 1958). Since NO has been shown to play an important role in the physiology of penile erection, it is not surprising that the aetiology of impotence has been linked to impaired nitrergic neurotransmission. In vitro and in vivo studies in animals and in human strongly suggest that a nitrergic defect may underlie the pathophysiology of erectile dysfunction (Saenz de Tejada et al., 1989; Azadzoi & Saenz de Tejada, 1992; Vernet et al., 1995; Rehman et al., 1997). The mechanism by which the nitrergic defect occurs in diabetes is, however, unknown. We have, therefore, investigated morphological and functional changes in nitrergic nerves in penile tissue from experimental streptozotocin-induced diabetic rats.

The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat.

Abstract: 1. We have studied the behavioural effects of intraplantar injections of adenosine 5'-triphosphate (ATP) and related compounds in freely moving rats and investigated whether these nociceptive effects are augmented in the presence of inflammatory mediators. 2. We find that in normal animals ATP and analogues produce dose-dependent nocifensive behaviour (seen as bursts of elevation of the treated hindpaw), and localized thermal hyperalgesia. The rank order of potency was: alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-methylene ATP) > 2-methylthioadenosine triphosphate (2-methylthio ATP) > ATP. After neonatal treatment with capsaicin, to destroy small calibre primary sensory neurones, nocifensive behaviour was largely absent. 3. The effects of ATP analogues were assessed in three models of peripheral sensitization: 2 h after dilute intraplantar carrageenan (0.25% w v(-1)); 24 h after irradiation of the hindpaw with ultraviolet (U.V.) B; immediately following prostaglandin E2 (PGE2) treatment. In all models the effect of alpha,beta-methylene ATP was greatly augmented. After carrageenan, significant hindpaw-lifting behaviour activity was induced by injection of only 0.05 nmol of alpha,beta-methylene ATP, some 100 times less than necessary in normal skin. 4. Our data suggest that it is much more likely that endogenous levels of ATP will reach levels capable of exciting nociceptors in inflamed versus normal skin. Our data also suggest the involvement of P2X3 receptor subunits in ATP-induced nociception.

Effects of the soluble guanylyl cyclase activator, YC-1, on vascular tone, cyclic GMP levels and phosphodiesterase activity.

Abstract: The vasomotor and cyclic GMP-elevating activity of YC-1, a novel NO-independent activator of soluble guanylyl cyclase (sGC), was studied in isolated rabbit aortic rings and compared to that of the NO donor compounds sodium nitroprusside (SNP) and NOC 18. Similarly to SNP and NOC 18, YC-1 (0.3–300 μM) caused a concentration-dependent, endothelium-independent relaxation that was greatly reduced by the sGC inhibitor 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ 10 μM; 59% inhibition of dilation induced by 100 μM YC-1) suggesting the activation of sGC as one mechanism of action. Preincubation with YC-1 (3 and 30 μM) significantly increased the maximal dilator responses mediated by endogenous NO in aortic rings that was released upon exposure to acetylcholine, and enhanced the dilator response to the exogenous NO-donors, SNP and NOC 18, by almost two orders of magnitude. Vasoactivity induced by SNP and YC-1 displayed different kinetics as evidenced by a long-lasting inhibition by YC-1 (300 μM) on the phenylephrine (PE)-induced contractile response, which was not fully reversible even after extensive washout (150 min) of YC-1, and was accompanied by a long-lasting elevation of intracellular cyclic GMP content. In contrast, SNP (30 μM) had no effect on the vasoconstrictor potency of PE, and increases in intravascular cyclic GMP levels were readily reversed after washout of this NO donor compound. Surprisingly, YC-1 not only activated sGC, but also affected cyclic GMP metabolism, as it inhibited both cyclic GMP break down in aortic extracts and the activity of phosphodiesterase isoforms 1–5 in vitro. In conclusion, YC-1 caused persistent elevation of intravascular cyclic GMP levels in vivo by activating sGC and inhibiting cyclic GMP break down. Thus, YC-1 is a highly effective vasodilator compound with a prolonged duration of action, and mechanisms that are unprecedented for any previously known sGC activator. British Journal of Pharmacology (1999) 127, 195–203; doi:10.1038/sj.bjp.0702495

Effects of vanadium complexes with organic ligands on glucose metabolism: a comparison study in diabetic rats

Abstract: 1 Vanadium compounds can mimic actions of insulin through alternative signalling pathways The effects of three organic vanadium compounds were studied in non-ketotic, streptozotocin-diabetic rats: vanadyl acetylacetonate (VAc), vanadyl 3-ethylacetylacetonate (VEt), and bis(maltolato)oxovanadium (VM) A simple inorganic vanadium salt, vanadyl sulphate (VS) was also studied 2 Oral administration of the three organic vanadium compounds (125 mg vanadium element 1(-1) in drinking fluids) for up to 3 months induced a faster and larger fall in glycemia (VAc being the most potent) than VS Glucosuria and tolerance to a glucose load were improved accordingly 3 Activities and mRNA levels of key glycolytic enzymes (glucokinase and L-type pyruvate kinase) which are suppressed in the diabetic liver, were restored by vanadium treatment The organic forms showed greater efficacy than VS, especially VAc 4 VAc rats exhibited the highest levels of plasma or tissue vanadium, most likely due to a greater intestinal absorption However, VAc retained its potency when given as a single ip injection to diabetic rats Moreover, there was no relationship between plasma or tissue vanadium levels and any parameters of glucose homeostasis and hepatic glucose metabolism Thus, these data suggest that differences in potency between compounds are due to differences in their insulin-like properties 5 There was no marked toxicity observed on hepatic or renal function However, diarrhoea occurred in 50% of rats chronically treated with VS, but not in those receiving the organic compounds 6 In conclusion, organic vanadium compounds, in particular VAc, correct the hyperglycemia and impaired hepatic glycolysis of diabetic rats more safely and potently than VS This is not simply due to improved intestinal absorption, indicating more potent insulin-like properties

Activation of nitric oxide synthase by β2‐adrenoceptors in human umbilical vein endothelium in vitro

Abstract: 1. Some animal studies suggest that beta-adrenoceptor-mediated vasorelaxation is in part mediated through nitric oxide (NO) release. Furthermore, in humans, we have recently shown that forearm blood flow is increased by infusion of beta2-adrenergic agonists into the brachial artery, and the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) inhibits this response. 2. The purpose of the present study was to determine whether stimulation of human umbilical vein endothelial beta-adrenoceptors causes vasorelaxation and nitric oxide generation, and whether this might be mediated by cyclic adenosine-3',5'-monophosphate (cyclic AMP). 3. Vasorelaxant responses were determined in umbilical vein rings to the nonselective beta-adrenergic agonist isoprenaline and to the cyclic AMP analogue dibutyryl cyclic AMP, following precontraction with prostaglandin F2alpha. 4. NOS activity was measured in cultured human umbilical vein endothelial cells (HUVEC) by the conversion of [3H]-L-arginine to [3H]-L-citrulline, and adenylyl cyclase activity by the conversion of [alpha-32P]-ATP to [32P]-cyclic AMP. 5. Isoprenaline relaxed umbilical vein rings, and this vasorelaxation was abolished by beta2- (but not beta1-) adrenergic blockage, and by endothelium removal or 1 mM L-NMMA. In addition, vasorelaxant responses to dibutyryl cyclic AMP were inhibited by 1 mM L-NMMA, with a reduction in Emax from 90.0+/-9.3% to 50.5+/-9.9% (P<0.05). 6. Isoprenaline 1 microM increased NOS activity in HUVEC (34.0+/-5.9% above basal, P<0.001). Furthermore, isoprenaline increased adenylyl cyclase activity in a concentration-dependent manner; this response was inhibited by beta2 (but not beta1-) adrenergic blockade. Forskolin 1 microM and dibutyryl cyclic AMP 1 mM each increased NOS activity in HUVEC, to a degree similar to isoprenaline 1 microM. The increase in L-arginine to L-citrulline conversion observed with each agent was abolished by coincubation with NOS inhibitors. 7. These results indicate that endothelial beta2-adrenergic stimulation and cyclic AMP elevation activate the L-arginine/NO system, and give rise to vasorelaxation, in human umbilical vein.

Protection by imidazol(ine) drugs and agmatine of glutamate‐induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor

Abstract: This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1–100 μM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 μM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μM (EC100) L-glutamate with the rank order (EC50 in μM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole] (101)>RX821002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10–12 μM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore. Keywords: Imidazoline, agmatine, neurotoxicity, neuroprotection, glutamate, N-methyl-D-aspartate receptors, cerebellar granule cells Introduction Several studies have demonstrated that imidazol(ine)/guanidine compounds display nanomolar affinities for various non-adrenoceptor sites, the so-called imidazoline receptors, and that the interaction with these recognition sites elicits various central and peripheral effects (for a review see Bousquet, 1995; French, 1995; Regunathan & Reis, 1996; Molderings, 1997). The amine agmatine (decarboxylated arginine) has been proposed as an endogenous agonist at imidazoline receptors (Li et al., 1994). Independently of their affinities on these receptors, imidazol(ine)/guanidine compounds also interact (in the micromolar range) with the monoamine oxidase enzymes (Ozaita et al., 1997) and various cation channels. These compounds block ATP-sensitive K+ channels (K+ATP channels) in pancreatic β-cells and rat insulinoma (RIN) cells and this leads to the stimulation of insulin release (Jonas et al., 1992; Olmos et al., 1994; Berdeu et al., 1997; Proks & Ashcroft, 1997). Imidazol(ine) drugs inhibit the acetylcholine-induced secretion of catecholamines in adrenal chromaffin cells (Ohara-Imaizumi & Kumakura, 1992) by blocking nicotinic acetylcholine receptors (Musgrave et al., 1995). These compounds also interact with 5-HT3 receptors in N1E-115 cells inhibiting the veratridine-induced influx of guanidinium to these cells (Molderings et al., 1996). Finally, an interaction with red cell Gardos channels (Coupry et al., 1996) and with rat brain N-methyl-D-aspartate (NMDA) receptors (Olmos et al., 1996) has also been demonstrated. Glutamate, the major excitatory neurotransmitter in the brain, is also a potent excitotoxin, inasmuch as prolonged exposure of most cultured neurones to micromolar concentrations of this aminoacid leads to neurotoxicity (Choi, 1988). In this sense, glutamate exposure of primary cultures of cerebellar granule cells is a well characterized model of neurotoxicity which results from the overstimulation of the NMDA receptor (Lysko et al., 1989; Schramm et al., 1990; Berman & Murray, 1996), leading to alterations in Ca2+ homeostasis (Milani et al., 1991). In this context, imidazol(ine) drugs and agmatine have been shown to be neuroprotective in brain injuries of necrotic (Gustafson et al., 1990; Maiese et al., 1992; Gilad et al., 1996) and apoptotic neuronal cell death (Olmos et al., 1999) in which glutamate-mediated neurotoxicity is clearly involved as the mechanism determining cell death. Thus, the present study was designed (1) to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells and (2) to seek whether imidazol(ine) drugs could exert neuroprotective effects through NMDA receptor blockade. A preliminary report of a portion of this study has been previously presented in abstract form (DeGregorio-Rocasolano et al., 1999).

Therapeutic index for rosiglitazone in dietary obese rats: separation of efficacy and haemodilution

Abstract: 1. The blood glucose-lowering efficacy of rosiglitazone (RSG) and the mechanisms of associated weight gain were determined in dietary obese rats (DIOs). DIO and chow-fed rats received RSG 0.3-30 mg kg-1 daily for 21 days. 2. In DIOs, plasma glucose and insulin concentrations were reduced by RSG at dosages of 3 and 10 mg kg-1, respectively. Homeostasis model assessment (HOMA) indicated the threshold for a reduction of insulin resistance was 1 mg kg-1. Neither glucose nor insulin levels were affected by treatment in chow-fed rats. 3. RSG 0.3 mg kg-1 lowered free fatty acids (FFAs) in DIOs, whereas for plasma triglycerides (TGs), the threshold was 3 mg kg-1. By contrast, the threshold for reducing packed red cell volume (PCV) and increasing cardiac mass was 10 mg kg-1. Thus, the therapeutic index for RSG in DIOs was >3 and 3 fold below the threshold for causing haemodilution and cardiac hypertrophy in DIO rats.

5‐hydroxytryptamine receptors mediating contraction in human small muscular pulmonary arteries: importance of the 5‐HT1B receptor

Abstract: 1. The 5-hydroxytryptamine (5-HT) receptors mediating vasoconstriction in isolated human small muscular pulmonary arteries (SMPAs) were determined using techniques of wire myography and reverse transcription-polymerase chain reaction (RT - PCR). 2. The agonists 5-HT, 5-carboxamidotryptamine (5-CT, unselective for 5-HT1 receptors) and sumatriptan (selective for 5-HT1B/D receptors) all caused contraction and were equipotent (pEC50s: 7.0+/-0.2, 7.1+/-0.3 and 6.7+/-0.1, respectively) suggesting the presence of a 5-HT1 receptor. 3. Ketanserin (5-HT2A-selective antagonist, 0.1 microM) inhibited 5-HT-induced contractions only at non-physiological/pathological concentrations of 5-HT (>0.1 microM) whilst GR55562 (5-HT1B/1D-selective antagonist, 1 microM) inhibited 5-HT-induced contractions at all concentrations of 5-HT (estimated pKB=7.7+/-0.2). SB-224289 (5-HT1B-selective antagonist, 0.2 microM) inhibited sumatriptan-induced contractions (estimated pKB=8.4+/-0.1) whilst these were unaffected by the 5-HT1D-selective antagonist BRL15572 (0.5 microM) suggesting that the 5-HT1B receptor mediates vasoconstriction in this vessel. 4. RT - PCR confirmed the presence of substantial amounts of mRNA for the 5-HT2A and 5-HT1B receptor subtypes in these arteries whilst only trace amounts of 5-HT1D receptor message were evident. 5. These findings suggest that a heterogeneous population of 5-HT2A and 5-HT1B receptors co-exist in human small muscular pulmonary arteries but that the 5-HT1B receptor mediates 5-HT-induced vasoconstriction at physiological and pathophysiological concentrations of 5-HT. These results have important implications for the treatment of pulmonary hypertension in which the 5-HT1B receptor may provide a novel and potentially important therapeutic target.