Benzamide potentiation of behavioral apomorphine-induced effects; mechanism involved

Summary A new N-pyridinyl benzamide was found to potentiate strongly the effects of apomorphine on the motility of reserpinized mice and on circling behavior. Since dopaminergic agonist activity could not account for this potentiation, involvement of-adrenergic agonist activity provided the only consistent explanation.Acknowledgments. We wish to thank Mr A. Decoodt for his excellent technical assistance and the Laboratoire de Chimie Therapeutique (Prof. Le Baut), Université de Nantes, for providing samples of N-PCB.     

The ecdysteroid agonist/antagonist and brassinosteroid-like activities of synthetic brassinosteroid/ecdysteroid hybrid molecules

A series of synthetic hybrid brassinosteroid/ecdysteroid structures has been assessed for their ecdysteroid agonist/antagonist activities in the Drosophila melanogaster B(II) cell bioassay and for brassinosteroid-like activity in the rice lamina inclination test. Most of the compounds proved inactive for ecdysteroid agonist activity, demonstrating the specificity of the ecdysteroid receptor for compounds closely structurally related to 20-hydroxyecdysone. However, compound 18, with 14alpha-hydroxy-7-en-6-one and 22S-hydroxy functionalities (as in most active ecdysteroids), possessed distinct agonist activity (median effective concentration = 1.4 x 10(-5) M), although this is still almost 2000-fold less active than 20-hydroxyecdysone (25). Compounds 13 and 15 possessed weak agonist activity. Compounds 5, 11 and 14 weakly antagonised the action of 20-hydroxyecdysone (at 5 x 10(-8) M) on B(II) cells. In the brassinosteroid bioassay, most of the tested compounds showed activity. This may reflect the metabolic capability of plant tissue to convert test compounds to more active analogues. However, it is clear that biological activity declines as the structure of the test compound deviates further from that of castasterone (16). Three ecdysteroids (25, 26 and 27) are completely inactive in the rice lamina inclination test. These studies demonstrate the high specificities of the insect ecdysteroid receptor and the plant brassinosteroid receptor and indicate that phytoecdysteroids, even in high concentrations, would not interfere with brassinosteroid signalling pathways in plants where the two classes of compounds co-occur. Equally, brassinosteroids would not interfere with ecdysteroid signalling in insects, especially if one takes into account the low concentrations of brassinosteroids in the diet of phytophagous insects.     

Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction

Platelet procoagulant activity is mainly determined by the extent of surface-exposed phosphatidylserine (PS), controlled by the activity of aminophospholipid translocase and phospholipid scramblase. Here, we studied both transport activities in single platelets upon stimulation with various agonists. Besides the formation of procoagulant microparticles, the results show that a distinct fraction of the platelets exposes PS when stimulated. The extent of PS exposure in these platelet fractions was similar to that in platelets challenged with Ca²⁺-ionophore, where all cells exhibit maximal attainable PS exposure. The size of the PS-exposing fraction depends on the agonist and is proportional to the platelet procoagulant activity. Scramblase activity was observed only in the PS-exposing platelet fraction, whereas translocase activity was exclusively detectable in the fraction that did not expose PS. We conclude that, irrespective of the agonist, procoagulant platelets exhibit maximal surface exposure of PS by switching on scramblase and inhibiting translocase activity.Received 8 March 2005; received after revision 19 April 2005; accepted 13 May 2005     

Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells

In this study, we examined agonist-induced internalization, recycling and signalling (measure of cAMP levels) of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by a receptor-binding assay on viable cells. The agonist nociceptin/orphanin FQ (NC) promoted rapid internalization of the hNOP receptor (approximately 78% of cell surface receptors were lost after 2 min exposure to 1 microM NC) in a clathrin- and ATP-dependent manner. Internalization was more rapid and marked in CHO-K1 cells than, as we previously reported, in SK-N-BE cells. This difference may be related to higher levels of beta-arrestin isoforms detected in CHO-K1 than in SK-N-BE cells. hNOP receptor internalization was partially reversible and recycling occurred in the presence of the agonist; receptor recycling was dependent on okadaic acid-sensitive phosphatases and was blocked by monensin. Confocal microscopy analysis confirmed the internalization and the recycling back to the plasma membrane of an epitope-tagged hNOP receptor expressed in CHO-K1 cells. These receptors underwent rapid desensitization upon agonist challenge: NC efficacy in inhibiting forskolin-stimulated cAMP production was significantly reduced 10 min after exposure and correlated with the rate of receptor internalization. Moreover, we observed that blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. Thus, the dynamic cycle between hNOP receptor activation, internalization and recycling determines the activity of this receptor on the cell surface.     

Beta-methyl carboline, a benzodiazepine inverse agonist, attenuates the effect of triazolam on the circadian rhythm of locomotor activity

The benzodiazepine triazolam, the benzodiazepine inverse agonist, beta-methyl carboline (beta-CCM) or both, were administered to adult male hamsters under conditions of constant light. When given alone, triazolam induced phase advances in the circadian activity rhythm of about 90 min, while beta-CCM when given alone, had no effect on phase of the activity rhythm. However, when triazolam and beta-CCM were given at the same time, the magnitude of the phase advances induced by triazolam were attenuated to about 30 min. These data, in conjunction with previous results, provide pharmacological evidence for a GABAergic system involved in the regulation of a central circadian pacemaker.     

Antidiuretic effects of oxytocin in the Brattleboro rat

The antidiuretic activity of oxytocin (OT) was measured in Brattleboro rats with congenital diabetes insipidus. A dose dependent antidiuretic response was found in animals receiving chronic infusions of 0.1 micrograms/h, 1.0 micrograms/h, and 5 micrograms/h of OT. OT infused at the rate of 5 micrograms/h over a 7-day period completely reversed the symptoms of diabetes insipidus. The results support the concept that OT serves as a weak agonist of vasopressin at the level of the kidney and at pharmacological levels exhibits antidiuretic activity.     

Prolactin and growth hormone releasing activity of [D-Met2, Pro5]-enkephalinamide in the rat after systemic administration

Summary The growth hormone (GH) and prolactin releasing (PRL) activity of [D-Met², Pro⁵]-enkephalinamide (EKNH₂), an opioid peptide analog with higher opiate agonist activity that morphine, was compared in the unanesthetized male rat to those of equimolar doses of morphine upon systemic injection. EKNH₂ proved to be a higher PRL, but not GH, releaser than the opiate alkaloid.     

Cellular models for the detection and evaluation of drugs that modulate human phagocyte activity

Simple testing models have been developed for the evaluation of chemical or biological compounds that modulate the activity of human phagocytes. Human neutrophils from buffy coats of donor blood are used. They are stimulated with receptor agonists, and the effects of test compounds on exocytosis of different enzymes, the generation of superoxide (respiratory burst), and cytotoxicity are quantified. All assays are performed in microtiter plates and the responses are evaluated by multi-well photometry or fluorimetry. The models are apt to detect compounds acting on phagocytes as agonists or antagonists, signal transduction activators or inhibitors and primers of agonist responses, and to assess cytotoxic effects.     

Cellular models for the detection and evaluation of drugs that modulate human phagocyte activity

Summary Simple testing models have been developed for the evaluation of chemical or biological compounds that modulate the activity of human phagocytes. Human neutrophils from buffy coats of donor blood are used. They are stimulated with receptor agonists, and the effects of test compounds on exocytosis of different enzymes, the generation of superoxide (respiratory burst), and cytotoxicity are quantified. All assays are performed in microtiter plates and the responses are evaluated by multi-well photometry or fluorimetry. The models are apt to detect compounds acting on phagocytes as agonists or antagonists, signal transduction activators or inhibitors and primers of agonist responses, and to assess cytotoxic effects.     

Stress and putative endogenous ligands for benzodiazepine receptors: the importance of characteristics of the aversive situation and of differential emotionality in experimental animals.

The relationships between anxiety/stress, possible endogenous ligands for benzodiazepine receptors and the behavioral modification by drugs are discussed in this short review, including the specific characteristics of elements involved in those interactions, e.g. ones concerning the aversiveness of the stressful situation and the nature of the organism under investigation. These are important factors when considering aversive tasks, insofar as they may involve stressful conditions which differ in intensity and in the degree of control afforded the subject. These characteristics may well lead to differing functional effects on GABA-gated chloride channels or, in other words, to an incongruous balance between endogenous benzodiazepine receptor agonist and inverse agonist activity. This is not surprising, as it is well known that different forms of stressors often actually produce divergent behavioral, physiological and biochemical effects. This review also illustrates the necessity of taking into account the variable effects of stressors and/or drugs on animals differing in reactivity or emotionality, even in the case of 'non-selected' stocks. The implication is made that, by genetic and/or environmental manipulation of the emotional state of the animals used, it will be possible to obtain more clearly definable results in neuropharmacological and psychopharmacological studies.     

Induction of rotational behaviour by intranigral baclofen suggests possible GABA-agonist activity

Summary In rats, unilateral injections of the GABA-derivative baclofen into the zona reticulata of the substantia nigra produced a contralateral rotation that was translated to ipsilateral rotation under the influence of amphetamine. These results mimic those following unilateral elevation of GABA levels in the substantia nigra and suggest that baclofen may have some GABA agonist activity following intracerebral injection.Baclofen was a generous gift of Ciba-Geigy Ltd.J. L. W. is a Medical Research Council Scholar.     

Effects of receptor agonists on polyamine concentrations and spermidine/spermine N1-acetyltransferase activity in rat parotid gland

The polyamine putrescine might be formed via a degradation (catalyzed by spermidine/spermine N¹-acetyltransferase, SSAT) of the higher polyamines spermidine and spermine to putrescine. The involvement of different intracellular signal pathways in the regulation of putrescine formation was studied in explants and in cultured cells of rat parotid glands by using receptor agonists that activate separate second messenger systems, and measuring their effects on the concentrations of putrescine, spermidine and spermine and on the SSAT activity. The -adrenoceptor agonist isoprenaline, which is an activator of cAMP formation, increased the putrescine concentration and stimulated the SSAT activity. Pilocarpine, a drug that activates the muscarinic receptors and thereby enhances the phosphoinositide turnover, had no effect on either the polyamine concentrations or on the SSAT activity. Epidermal growth factor (EGF), which induces activation of a protein tyrosine kinase, had no effect on the polyamine concentrations or on the SSAT activity. The adenylate cyclase activator forskolin increased the glandular levels of putrescine. Taken together, these findings suggest that increases in putrescine concentration in cultured rat parotid gland cells are accompanied by accumulation of cAMP.     

GABA-A-mediated gastrin release induced by baclofen in the isolated vascularly perfused rat stomach

Summary In order to investigate the role of peripheral GABA-B receptors, the effects of the putative GABA-B agonist baclofen on immunoreactive gastrin release from an isolated vascularly perfused rat stomach preparation were examined. The vascular infusion of baclofen at graded concentrations induced a dose-dependent increase in gastrin release; this was unaffected by the GABA-B antagonist delta-aminovaleric acid, but was fully prevented by the selective GABA-A antagonist bicuculline as well as by atropine or tetrodotoxin. These results suggest that the stimulant effects of baclofen are mediated by nervous cholinergic structures, associated with GABA-A receptors, and indicate that this GABA-B agonist must be regarded as a partial agonist of peripheral GABA-A receptors.     

GABA-A-mediated gastrin release induced by baclofen in the isolated vascularly perfused rat stomach

In order to investigate the role of peripheral GABA-B receptors, the effects of the putative GABA-B agonist baclofen on immunoreactive gastrin release from an isolated vascularly perfused rat stomach preparation were examined. The vascular infusion of baclofen at graded concentrations induced a dose-dependent increase in gastrin release; this was unaffected by the GABA-B antagonist delta-aminovaleric acid, but was fully prevented by the selective GABA-A antagonist bicuculline as well as by atropine or tetrodotoxin. These results suggest that the stimulant effects of baclofen are mediated by nervous cholinergic structures associated with GABA-A receptors, and indicate that this GABA-B agonist must be regarded as a partial agonist of peripheral GABA-A receptors.     

A gonadotropin releasing hormone analog induces spermiation in intact and hypophysectomized frogs, Rana esculenta

The sperm-releasing activity of a gonadotropin releasing hormone (GnRH) agonist, Buserelin (GnRH) and hypophysis homogenate (PD) preparations was studied in intact and hypophysectomized (PDX) frogs, Rana esculenta. In addition, human chorion gonadotropin (hCG) was tested in PDX animals, and GnRH antagonist (GnRHA) treatments were carried out in intact and PDX animals, in combination with the hormonal injections. GnRH or PD treatments were able to elicit spermiation in intact and PDX animals. While GnRH, injected 24 h later, was again effective in inducing spermiation in intact animals, this was not the case in PDX frogs. GnRHA counteracted GnRH effects in intact frogs. Moreover, in PDX animals GnRHA injections counteracted the sperm-releasing activity induced by hCG or GnRH, but failed to inhibit sperm-releasing activity induced by PD homogenate.     

Evaluation of monoaminergic receptors in the genetically epilepsy prone rat

The intensity of sound-induced convulsions in the genetically epilepsy-prone rat (GEPR) was reduced in a dose related fashion by intracerebroventricular administration of dobutamine, (beta 1 agonist), terbutaline (beta 2 agonist) or phenylephrine (alpha 1 agonist). BHT-920 (alpha 2 agonist) did not cause a dose-related decrease in sound-induced convulsion intensity. Binding studies showed that whole brain alpha and beta receptor densities (Bmax) were normal while the Kd was increased for the beta ligand in GEPR brain.     

Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion

Transients in myoplasmic [Ca2+] and in phosphorylation of the 20,000 dalton light chain of myosin have been reported following stimulation of vascular smooth muscle by various agonists. Since these transients are rapid compared with the time required to attain a steady-state stress, agonist diffusion rates may be a significant limitation in activation. The purpose of this study was to estimate the effect of agonist diffusion rates on the time course of activation as assessed by mechanical measurements of stress development and isotonic shortening velocities and by determinations of the time course of myosin phosphorylation. The approach was to measure these parameters in K+ -stimulated preparations of the swine carotid media of varying thicknesses and to estimate the theoretical contributions imposed by diffusion rates and the presence of a diffusion boundary layer surrounding the tissue. The results show that the time course of parameters which are tissue averages such as stiffness, active stress, and myosin phosphorylation is dominated by agonist diffusion rates. The sequence of events involved in excitation-contraction coupling including agonist actions on the cell membrane, Ca2+ release, activation of myosin light chain kinase, and cross-bridge phosphorylation appear to be very rapid events compared with stress development. Estimates of unloaded or lightly loaded shortening velocities which are not simple tissue averages appear to provide an improved estimate of activation rates.     

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.     

Stress and putative endogenous ligands for benzodiazepine receptors: The importance of characteristics of the aversive situation and of differential emotionality in experimental animals

The relationships between anxiety/stress, possible endogenous ligands for benzodiazepine receptors and the behavioral modification by drugs are discussed in this short review, including the specific characteristics of elements involved in those interactions, e.g. ones concerning the aversiveness of the stressful situation and the nature of the organism under investigation. These are important factors when considering aversive tasks, insofar as they may involve stressful conditions which differ in intensity and in the degree of control afforded the subject. These characteristics may well lead to differing functional effects on GABA-gated chloride channels or, in other words, to an incongruous balance between endogenous benzodiazepine receptor agonist and inverse agonist activity. This is not surprising, as it is well known that different forms of stressors often actually produce divergent behavioral, physiological and biochemical effects. This review also illustrates the necessity of taking into account the variable effects of stressors and/or drugs on animals differing in reactivity or emotionality, even in the case of non-selected stocks. The implication is made that, by genetic and/or environmental manipulation of the emotional state of the animals used, it will be possible to obtain more clearly definable results in neuropharmacological and psychopharmacological studies.     

Potentiating effect of phenylephrine on isoproterenol activation of thyroxine type II deiodinase in the pineal gland of adult rats

In the present study we show, for the first time, that phenylephrine (PHE), an -adrenergic receptor agonist, potentiates the effect of isoproterenol (ISO), a -adrenergic agonist, in activating pineal type II5-deiodinase (5-D) activity. The potentiating effect of PHE was observed only at doses of ISO which induce submaximal activation of the enzyme. However, at doses which lead to maximal activation of the enzyme, PHE was ineffective. The results suggest that not only -, but also -adrenergic receptors, are involved in the sympathetic noradrenergic regulation of pineal 5-D activity in the adult rat.