PubMed Health⌕ Search

Biomedical subjects

P M Laduron

Publications and source records attributed to P M Laduron.

At least 91 records · Page 5Linked to original sources
PubMed1985

Solubilization of rat brain serotonin-S2 receptors using CHAPS/salt.

Serotonin-S2 receptors from rat frontal cortex were solubilized with a mixture of 6.8 mM CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate) and 1.4 M sodium chloride. This new solubilization procedure solubilized about 40% of the membrane-bound serotonin-S2 receptors in an active form. The solubilized receptors were not sedimented after 1 h of centrifugation at 100 000 X g and they passed freely through 0.20 micron filters. The solubilized preparation showed high affinity binding of [3H]ketanserin and revealed a typical serotonin-S2 receptor profile: binding could be displaced by nanomolar concentrations of different serotonin antagonists and by micromolar concentrations of serotonin agonists. Compounds belonging to other pharmacological classes were poorly, or not active. Upon density gradient sedimentation, Svedberg coefficients of approximately 5 S were found on sucrose gradients made with H2O or D2O as the solvent. This was much lower than the value of 11.5 S previously reported from lysophosphatidylcholine-solubilized serotonin-S2 receptors.

Animals↗
PubMed1985

Photoaffinity probes for serotonin and histamine receptors. Synthesis and characterization of two azide analogues of ketanserin.

Two azide analogues of ketanserin (6- and 7-azido-3-[2- [4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-2, 4(1H,3H)-quinazolinedione) were synthesized and tested as possible photoaffinity probes for serotonin-S2 and histamine-H1 receptors. In reversible binding experiments, the azides showed high affinity for both receptor types. When membrane preparations were incubated with nanomolar concentrations of 7-azidoketanserin and subsequently irradiated with UV light, both serotonin and histamine receptors became irreversibly blocked. This irreversible binding was dependent on azide concentrations and time of irradiation and did not change in the presence of the scavenger p-aminobenzoic acid. In contrast, irreversible blockade at low concentrations of 6-azidoketanserin was only obtained for histamine receptors. However, this blockade was abolished by addition of the scavenger p-aminobenzoic acid indicating that it was not due to a real photoaffinity mechanism. In the rat prefrontal cortex, irreversible blocking of serotonin receptors with 7-azidoketanserin could be inhibited by serotonin agonists or antagonists but not by histaminergic compounds. On the contrary, in the guinea pig cerebellum, inactivation of histamine receptors could be inhibited by histamine antagonists and histamine itself but not by serotonergic compounds. This provides a way for differential photolabeling of either of these receptors.

4-Aminobenzoic Acid↗
PubMed1985

Retrograde axonal transport of receptor-bound opiate in the vagus and delayed accumulation in the nodose ganglion.

Opiate receptors measured in vivo with [3H]lofentanil in the rat vagus nerve were found to accumulate on both sides of a ligature. The time-course of accumulation was completely different in the proximal and the distal segments; the labelling was maximal 4 h after injection of [3H]lofentanil above the ligature but 16-24 h below the ligature. In unligated rats, a peak of radioactivity appeared in the nodose ganglion 16 h after injection; vagotomy, vinblastine or chronic treatment with capsaicin prevented the appearance of this delayed accumulation in the ganglion. These foregoing experiments suggest that opiate may act in the cell body of sensory neurones after being internalized at the nerve terminals and then transported retrogradely through fast axoplasmic mechanisms.

Animals↗
PubMed1985

Differential photoaffinity labelling of serotonin-S2 receptors and histamine-H1 receptors using 7-azidoketanserin.

7-Azidoketanserin, a potent photoaffinity probe for serotonin-S2 receptors was shown to irreversibly photoinactivate histamine-H1 receptors as well. The photolabelling of H1-receptors could be prevented by several selective histamine-H1 antagonists. In guinea pig cerebellum, a brain area that is highly enriched in H1-receptors, photolabelling could be directed exclusively to these receptors by adding a high concentration of pipamperone, which selectively blocks serotonin-S2 receptors. In rat pre-frontal cortex, a region that is enriched in serotonin-S2 receptors, pyrilamine was used to block H1-receptors, thereby directing the photolabelling exclusively to S2-receptors.

Affinity Labels↗
PubMed1985

Opiate receptors in brain labelled in vivo with [3H]lofentanil.

The in vivo binding of [3H]lofentanil was studied in various regions of the brain in rat. After intravenous injection of [3H]lofentanil the disposition of the labelled drug in the brain paralleled exactly the regional distribution of opiate receptors measured in in vitro binding assays. The labelling was saturable and could be prevented by naloxone when given before [3H]lofentanil, in all the regions except in the cerebellum. The long-lasting occurrence of the specific labelling was entirely compatible with the extremely slow dissociation rate of lofentanil and its long duration of action. This explains why [3H]lofentanil is not displaceable by naloxone in vivo. Subcellular fractionation experiments revealed that all the labelling in the frontal cortex but not in the cerebellum was particulate-bound and entirely displaceable by naloxone. The advantages of [3H]lofentanil in vivo are its extremely low non-specific binding and its ability to reveal very low occupancy of opiate receptors in brain.

Analgesics, Opioid↗
PubMed1985

Receptor-binding properties in vitro and in vivo of ritanserin: A very potent and long acting serotonin-S2 antagonist.

In vitro and in vivo receptor-binding properties of the new serotonin antagonist, ritanserin, are reported. In in vitro binding assays, ritanserin shows high affinity binding to serotonin-S2 sites in rat frontal cortex tissue: IC50 = 0.9 nM without drug preincubation and 0.3 nM with 30-min drug preincubation; IC50 values for histamine-H1, dopamine-D2, and adrenergic-alpha 1 and -alpha 2 sites were 39-, 77-, 107-, and 166-fold higher, and at up to 1 microM, the drug did not bind to serotonin-S1 sites. In in vitro assays, ritanserin dissociated very slowly from serotonin-S2 (t1/2 = 160 min) and histamine-H1 sites (t1/2 = 77 min) and rapidly from dopamine-D2 sites (t1/2 = 11 min). Half-times of dissociation from adrenergic-alpha 1 and -alpha 2 sites were 18 and 26 min. The inhibition by ritanserin of [3H]ketanserin binding was found to be partially noncompetitive and the inhibitory potency increased with drug preincubation. Due to the slow dissociation of ritanserin from the serotonin-S2 sites, the drug cannot be displaced completely by [3H]ketanserin. In contrast, inhibition by ritanserin of [3H]haloperidol binding to dopamine-D2 sites in rat striatum was fully competitive, in agreement with the rapid dissociation of the drug from the latter sites. In ex vivo binding assays using brain areas of rats and guinea pigs treated subcutaneously with ritanserin, occupation of serotonin-S2 sites was observed at very low dosage (50% occupation at 0.08-0.1 mg/kg) and sites remained occupied during a prolonged time period (greater than 70% occupation up to 48 hr after 2.5 mg/kg ritanserin). Histamine-H1 receptor sites in guinea pig cerebellum became occupied at dosages 25-fold higher than the dosage producing occupation of frontal cortical serotonin-S2 sites. Dopamine-D2 sites in rat striatum and cortical adrenergic-alpha 1 sites became only slightly occupied (less than 20%) at higher dosages and the effect was not dose-dependent. Adrenergic-alpha 2 sites were not occupied up to doses of 160 mg/kg given subcutaneously. In vivo binding assays using [3H]spiperone confirmed the occupation of frontal cortical serotonin-S2 sites following low dosage of ritanserin and a minor occupation of striatal dopamine-D2 sites. Levels of dopamine and serotonin and their metabolites remained unchanged in brain areas of rats orally treated with ritanserin up to dosages of 40 mg/kg. At 160 mg/kg, there seemed to be a slight reduction in dopamine and serotonin content.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Characterization of cholate-solubilized dopamine receptors from human, dog and rat brain.

[3H]Spiperone binding sites were solubilized in high yield from human, dog and rat brain with a mixture of sodium cholate (0.3% w/v) and sodium chloride (1.4 M). The binding sites were not sedimented after one hour at 100,000 g, they passed freely through 0.20 micron filters, migrated as a single peak in gradient sedimentation and were retarded upon gel filtration, proving that they were truly solubilized. The solubilized binding sites were definitely of dopaminergic nature. They showed saturable, reversible, high affinity binding of [3H]spiperone; displacement of [3H]spiperone binding by nanomolar concentrations of dopamine antagonists and micromolar concentrations of serotonin antagonists; stereo-specificity and a good correlation with drug affinities for membrane preparations. The non-displaceable, non-specific [3H]spiperone binding was very low. Gradient sedimentation analysis revealed a sedimentation coefficient of 12 S for dog solubilized preparations, 9 S for rat solubilized preparations and only 2.5 S for human solubilized preparations (values, uncorrected for detergent binding). Gel filtration experiments seem to confirm these molecular characteristics. Therefore the present results show that the dopamine receptor reveals the same pharmacological properties when solubilized with cholate-salt from rat, dog or human brain, while physico-chemical properties seem to indicate some differences.

Animals↗
PubMed1984

Photoaffinity labelling of dopamine receptors. Synthesis and binding characteristics of azapride.

A new compound, azapride, i.e. 4-azido-5-chloro-2-methoxy-N-[1-(phenylmethyl)-4-piperidinyl]benza mide, the azide derivative of the dopamine antagonist clebopride, was synthesized and tested for its usefulness as a photoaffinity probe for dopamine receptors. Without photolysis the azide behaved as a very selective dopamine antagonist. When a microsomal preparation of dog striatum was pretreated with the azide and subsequently irradiated with ultraviolet light, dopamine receptors became irreversibly blocked. This reaction was dependent on both azide concentration and time of irradiation. The irradiation-induced inhibition was quite selective for the dopamine receptor; various other receptor systems were not affected. Moreover the irreversible inhibition of dopamine receptors from dog striatum could be prevented by dopamine antagonists as well as by a dopamine agonist, but not by a serotonin antagonist. The irradiation-induced inactivation by azapride remained unchanged in the presence of the scavenger p-aminobenzoic acid. These findings provide evidence that azapride labels dopamine receptors specifically and irreversibly and by a true photoaffinity mechanism. It should therefore be of great value for further molecular characterization and purification of dopamine receptors.

Affinity Labels↗
PubMed1984

Solubilization of serotonin S2-receptors from human brain.

Serotonin S2-receptors were solubilized from human brain by means of the mild detergent, lysolecithin. Previous studies have shown that the serotonin S2-receptors in human brain are mainly enriched in the cortex. A total particulate fraction from human cortex was treated with 0.25% lysolecithin. [3H]Ketanserin binding sites from the soluble extract showed the binding characteristics of S2-receptors: potent 5HT antagonists like pirenperone, methysergide and pipamperone competed for [3H]ketanserin binding at nanomolar concentrations. The agonists bufotenin and serotonin themselves were more active than the potent dopamine agonist tetraline. Binding was saturable with a low KD (1.07 nM) and reversible. There was a good correlation between the drug potencies in both soluble and membrane preparations and also with the IC50 values previously obtained in membrane preparations and soluble extract from rat brain. Therefore, lysolecithin allows serotonin S2-receptors from human brain to be obtained in a molecularly dispersed form with the same high affinity properties as in the original membranes.

Animals↗
PubMed1984

Criteria for receptor sites in binding studies.

The study of binding is not an easy task especially because of the difficulty of interpreting the results in terms of binding on specific receptor sites. The problem is not new; what is new is the increasing amount of fanciful interpretation that such a technique has generated. The tendency to interpret anomalous or intriguing results in terms of new receptor subtypes seems to have reached its peak. The need to apply severe criteria becomes imperative before concluding that a binding site may be called a receptor site. Some pitfalls in binding studies will be discussed.

Animals↗
PubMed1984

Axonal transport of receptors: coexistence with neurotransmitter and recycling.

Receptor sites move along the axon of neurones by means of fast transport mechanisms. In rat sciatic nerves, the accumulation of muscarinic receptors above a ligature was linear up to 24 hr after the operation and exactly paralleled that of dopamine-beta-hydroxylase. After 6-hydroxydopamine treatment, there was a marked reduction of these receptor sites in the rat sciatic nerve and the spleen. Fractionation of dog splenic nerves by differential and isopycnic centrifugation enabled us to show that the muscarinic receptors in sympathetic nerves are associated with noradrenaline and dopamine-beta-hydroxylase in synaptic vesicles. Receptor and neurotransmitter may thus coexist in the same subcellular organelle. Opiate receptors measured in vitro and in vivo with 3H-lofentanil in the rat vagus nerve were found to accumulate on both sides of the ligature. After capsaicin treatment this accumulation was markedly reduced suggesting that the opiate receptors in the vagus are mainly associated with substance P neurones. Double ligature experiments suggest the existence of a recycling phenomenon in the perikaryon for vesicles containing muscarinic receptors and dopamine-beta-hydroxylase. This may represent a pathway to convey informational molecules from the extracellular to the intracellular compartment of a neurone.

Animals↗
PubMed1984

Axonal transport of opiate receptors in capsaicin-sensitive neurones.

Opiate receptors measured in vitro or in vivo with [3H]lofentanil in the rat vagus nerve were found to accumulate on both sides of a ligature, thus indicating a bidirectional axoplasmic transport of these receptors. When rats were treated with capsaicin, the accumulation of opiate receptors was tremendously reduced in the vagus whereas muscarinic receptors in ligated sciatic nerves were unaffected. Since capsaicin is known to affect sensory neurones, mostly those containing substance P, the present results support the idea that the opiate receptors in the vagus are associated with substance P neurones.

Animals↗
PubMed1984

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-beta-hydroxylase.

Presynaptic muscarinic receptors labeled with [3H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [3H]quinuclidinyl benzilate or [3H]dexetimide binding revealed a distribution profile similar to that of dopamine-beta-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-beta-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-beta-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.

Animals↗
PubMed1984

Regional and cortical laminar distributions of serotonin S2, benzodiazepine, muscarinic, and dopamine D2 receptors in human brain.

Serotonin S2, benzodiazepine, and muscarinic receptors showed different regional distributions in the human brain but were present in all cortical areas. The laminar distributions of [3H]ketanserin, [3H]diazepam, and [3H]quinuclidinylbenzilate were investigated in the temporal cortex and revealed a high density in the IIIrd and IVth layers. Dopamine D2 receptors were not detected in the cortex.

Adult↗