Andre Convents

High affinity binding of 3H rauwolscine and 3H RX781094 to α2 adrenergic receptors and non-stereo selective sites in human and rabbit brain cortex membranes

The radiolabeled antagonists 3H RX 781094 and 3H rauwolscine bind with high affinity to alpha 2 adrenergic receptors as well as to non-receptor sites in human and rabbit brain cortex membranes. These non-receptor sites form an important contaminant of the specific binding when non-specific binding is determined in the presence of 10 microM phentolamine or more. While phentolamine is no suitable ligand to discriminate both sites, (-)-epinephrine displays a sufficient affinity ratio to separate radioligand binding to these sites. When 1 microM (-)-epinephrine is used for the determination of the non-specific binding, both radioligands bind specifically to alpha 2 receptors. Under these conditions, 3H rauwolscine and 3H RX 781094 bind to the same amount of non-cooperative sites; binding isotherms for human brain are Bmax = 113 +/- 15 fmol/mg protein and Kd = 22.8 +/- 4.2 nM for 3H RX781094 and Bmax = 110 +/- 17 fmol/mg protein and Kd = 4.7 +/- 2.5 nM for 3H rauwolscine. Competition binding experiments show, for both radioligands and in both species, the typical pharmacological potency order of alpha 2 adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin for the antagonists and UK 14304 greater than p-aminoclonidine greater than or equal to (-)-epinephrine greater than (+)-epinephrine greater than isoproterenol for the agonists. Whereas the alpha 2 receptor sites display high affinity and stereoselectivity towards (-)-epinephrine and (+)-epinephrine, the non-receptor sites bind both epinephrine isomers with equal low affinity. Specific binding of both radioligands to these sites can be determined when total binding is performed in the presence of 1 microM (-)-epinephrine and non-specific binding the presence of 1 mM phentolamine. 3H rauwolscine binding to the non-stereoselective sites can be displaced with high affinity by 5-HT, suggesting binding to a 5-HT1-receptor. The 3H RX 781094 binding displays low affinity for most alpha adrenergic ligands and do not correspond to beta adrenergic, dopaminergic or serotonergic receptors.

Autoradiographic distribution of α2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [3H]idazoxan and [3H]rauwolscine

The regional distribution of [3H]idazoxan and [3H]rauwolscine was studied autoradiographically in human brain. [3H]Idazoxan binds with high affinity to alpha 2 adrenoceptors as well as to non-adrenergic sites (NAIBS). [3H]Rauwolscine, besides binding to alpha 2 adrenoceptors, also binds to 5-HT1A receptors. Both radioligands labelled the same population of alpha 2 adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of alpha 2 adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no alpha 2 adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable alpha 2 adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT1A receptors labelled by [3H]rauwolscine is in agreement with previous studies using [3H]8-OH-DPAT.

[3H]Rauwolscine labels α2-adrenoceptors and 5-HT1A receptors in human cerebral cortex

Abstract [ 3 H]Rauwolscine binds with high affinity to α 2 -adrenoceptors (K d = 4.8 ± 1.3 nM, B max = 79 ± 26 fmol/mg protein, micrmolar affinity for 5-HT) as well as to 5-HT 1 -like receptors (K d = 13 ± 2.7 nM, B max = 147 ± 11.4 fmol/mg protein, nanomolar affinity for 5-HT) in human brain cortex membranes. The K i values of 11 serotonergic compounds for the latter receptors agreed closely with those previously reported for 5-HT 1A sites but not with those for 5-HT 1B , 5-HT 1C and 5-HT 1D sites.

Prehospital Unassisted Assessment of Stroke Severity Using Telemedicine A Feasibility Study

Background and Purpose— We evaluated the feasibility and the reliability of remote stroke severity quantification in the prehospital setting using the Unassisted TeleStroke Scale (UTSS) via a telestroke ambulance system and a fourth-generation mobile network. Methods— The technical feasibility and the reliability of the UTSS were studied in healthy volunteers mimicking 41 stroke syndromes during ambulance transportation. Results— Except for 1 issue, high-quality telestroke assessment was feasible in all scenarios. The mean examination time for the UTSS was 3.1 minutes (SD, 0.4). The UTSS showed excellent intrarater and interrater variability (&rgr;=0.98 and 0.97; P<0.001), as well as excellent internal consistency and rater agreement. Adequate concurrent validity can be derived from the strong correlation between the UTSS and the National Institutes of Health Stroke Scale (&rgr;=0.90; P<0.001). Conclusions— Remote assessment of stroke severity in fast-moving ambulances using a system dedicated to prehospital telemedicine, 4G technology, and the UTSS is feasible and reliable.

Feasibility of AmbulanCe-Based Telemedicine (FACT) Study: Safety, Feasibility and Reliability of Third Generation In-Ambulance Telemedicine

Background Telemedicine is currently mainly applied as an in-hospital service, but this technology also holds potential to improve emergency care in the prehospital arena. We report on the safety, feasibility and reliability of in-ambulance teleconsultation using a telemedicine system of the third generation. Methods A routine ambulance was equipped with a system for real-time bidirectional audio-video communication, automated transmission of vital parameters, glycemia and electronic patient identification. All patients ( ≥18 years) transported during emergency missions by a Prehospital Intervention Team of the Universitair Ziekenhuis Brussel were eligible for inclusion. To guarantee mobility and to facilitate 24/7 availability, the teleconsultants used lightweight laptop computers to access a dedicated telemedicine platform, which also provided functionalities for neurological assessment, electronic reporting and prehospital notification of the in-hospital team. Key registrations included any safety issue, mobile connectivity, communication of patient information, audiovisual quality, user-friendliness and accuracy of the prehospital diagnosis. Results Prehospital teleconsultation was obtained in 41 out of 43 cases (95.3%). The success rates for communication of blood pressure, heart rate, blood oxygen saturation, glycemia, and electronic patient identification were 78.7%, 84.8%, 80.6%, 64.0%, and 84.2%. A preliminary prehospital diagnosis was formulated in 90.2%, with satisfactory agreement with final in-hospital diagnoses. Communication of a prehospital report to the in-hospital team was successful in 94.7% and prenotification of the in-hospital team via SMS in 90.2%. Failures resulted mainly from limited mobile connectivity and to a lesser extent from software, hardware or human error. The user acceptance was high. Conclusions Ambulance-based telemedicine of the third generation is safe, feasible and reliable but further research and development, especially with regard to high speed broadband access, is needed before this approach can be implemented in daily practice.

D2 dopamine receptors in calf globus pallidus: agonist high- and low-affinity sites not regulated by guanine nucleotide

Abstract: By use of the radioligand [3H]spiroperidol, D2 3,4‐dihydroxyphenylethylamine (dopamine) receptor binding characteristics were studied in calf globus pallidus and compared with those of neostriatum. Antagonist competition curves were monophasic and revealed similar affinities for neostriatum and globus pallidus, suggesting a uniform receptor population with one affinity state for antagonists. In both regions, competition curves with the agonist dopamine were biphasic, distinguishing a high‐ and low‐agonist‐affinity state. In neostriatum and globus pallidus, respectively, 45% and 19% of [3H]spiroperidol binding was displaced with high affinity and the remainder with low affinity. In neostriatum, the addition of 0.4 mM GTP resulted in a partial conversion from high‐ to low‐affinity state with a remaining highaffinity component of 15%. In globus pallidus, dopamine binding was not altered by GTP. The capability of GTP to modulate agonist binding to D2 receptors appears to be dependent on their neuroanatomical localization.

Characterization of alpha2-adrenergic receptors of calf retina membranes by [3H]-rauwolscine and [3H]-RX 781094 binding

Alpha 2-adrenergic receptors were identified in calf retina membranes by binding of the radiolabelled antagonists [3H]-RX 781094 and [3H]-rauwolscine. When 10 microM phentolamine was used to determine the non-specific binding, both radioligands labelled a single class of non-cooperative sites: Bmax = 1051 +/- 252 fmol/mg protein, Kd = 5.1 +/- 1.5 nM for [3H]-RX 78104 and Bmax = 1167 +/- 449 fmol/mg protein, Kd = 21.0 +/- 4.1 nM for [3H]-rauwolscine. Competition binding experiments showed the typical pharmacological potency order of alpha 2-adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin. Agonist competition binding curves revealed the presence of two receptor populations, having respectively high affinity (70% of the total receptor population) and low affinity for agonists, but with the same affinity for the antagonists. The high affinity sites could be converted into low affinity sites by guanine nucleotides. The non-specific binding of [3H]-RX 781094 was the same if 0.1 mM (-)-epinephrine was used instead of phentolamine. In contrast, the non-specific binding of [3H]-rauwolscine was markedly lower with (-)-epinephrine than with phentolamine. Under this condition, the Scatchard plot of [3H]-rauwolscine saturation binding was curvilinear, indicating the presence of low affinity sites for the radioligand in addition to alpha 2-adrenergic receptors. Competition binding experiments revealed that these low affinity sites were distinct from adrenergic receptors: the catecholamine agonists (-)- and (-)-epinephrine, (-)-norepinephrine, (-)-isoproterenol and dopamine competed with similar Ki values (microM range) whereas clonidine did not interact. Furthermore, these sites bound reserpine and the alpha 2-adrenergic antagonists yohimbine and rauwolscine but not phentolamine.

Conus venom interaction with α2-adrenergic receptors in calf retina membranes

?(2)-Adrenergic receptors were identified in calf retina membranes by the specific binding of the radiolabelled antagonist [(3)H]RX 781094. Crude venoms from various Conus species did not interact with the radioligand but were able to inhibit radioligand binding to the ?(2)-receptors with the following order of potency: C. planorbis (IC(50) = 2.1 ?g protein/ml) ? C. tessulatus (IC(50) = 2.7) >C. eburneus (IC(50) = 19) >C. textile (IC(50) = 54) >C. geographus (IC(50) = 130). Venom from 17 other species was less or not active at all. Venom competition binding curves were steep and not affected by GTP. In contrast, the ( ? )-epinephrine competition binding curve was shallow and underwent a rightward shift and steepening in the presence of GTP. The venom-?(2)-receptor interaction was completely inhibited by the calcium chelating reagent EGTA. These data suggest that the venom of certain Conus species contains peptide toxins which are capable of shielding the binding site of ?(2)-receptors in an antagonistic manner.

Subtypes of adrenergic and dopaminergic receptors in bovine cerebral blood vessels

Binding of the radiolabelled antagonists [3H]rauwolscine, [3H]SCH 23390 and (-)-[3H]dihydroalprenolol revealed the presence of alpha 2-adrenergic greater than DA1 dopaminergic greater than beta-adrenergic receptors in membrane preparations of calf basal cerebral arteries (basilar artery and circle of Willis) and pial vessels of the cerebral convexity. Computer-assisted analysis of ICI 118 551/(-)-[3H]dihydroalprenolol competition binding curves indicated the existence of beta 1- and beta 2-adrenergic receptor subtypes (beta 2/beta 1 ratio 7:3). No specific binding of [3H]prazosin (to alpha 1-adrenergic receptors) and [3H]spiroperidol (to DA2 dopaminergic receptors) was detected. Whereas DA1, beta 1- and beta 2-receptor densities were very similar in both blood vessel types, the alpha 2-receptor density was 3-fold higher in the pial vessels of the convexity. This suggests a functionally more important vasoconstrictor adrenergic control of the cerebral circulation in pial vessels of the convexity than in the arteries at the base of the brain.

Glycoprotein nature of α2-adrenergic receptors labeled with p-azido[3H] clonidine in calf retina membranes

α2,‐Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated agonist p‐azido[3H]clonidine. Saturation binding in the dark occurs with high affinity (1.3 ± 0.3 nM) to a single class of sites (1122 ± 67 fmol/mg protein). Irradiation of the membrane‐bound radioligand results in the labeling of a peptide band with an apparent size of 65 kDa and a characteristic pharmacological profile for an α2‐adrenergic receptor. The carbohydrate moieties of the α2‐receptor are characterized by lectin affinity chromatography and glycosidase treatment. The Nonidet P‐40‐solubilized, p‐azido[3H]clonidine‐labeled receptors are completely retained by Con A‐ as well as WGA‐Sepharose columns. Neuraminidase, α‐mannosidase and TFMS do not affect the electrophoretic mobility of the receptor on SDS‐PAGE whereas endoglycosidase F reduces the apparent size to 45 kDa.