Adrenergic Sites Research Articles

Pharmacokinetics, Safety and Tolerability of Rotigotine Transdermal Patch in Healthy Japanese and Caucasian Subjects

Background and ObjectivesRotigotine is a dopamine receptor agonist with activity across the D1 through to D5 receptors as well as select serotonergic and adrenergic sites; continuous transdermal delivery of rotigotine with replacement of the patch once daily maintains stable plasma concentrations over 24 h. Rotigotine is indicated for the treatment of early and advanced-stage Parkinson’s disease and moderate-to-severe idiopathic restless legs syndrome. The pharmacokinetics and pharmacodynamics of a drug may vary between subjects of different ethnic origin. This study evaluated the pharmacokinetics, safety, and tolerability of single-dose treatment with rotigotine transdermal patch in Japanese and Caucasian subjects.MethodsIn this open-label, parallel-group study, healthy male and female subjects of Japanese or Caucasian ethnic origin were matched by sex, body mass index, and age. A single transdermal patch delivering 2 mg/24 h rotigotine (patch content 4.5 mg) was applied to the ventral/lateral abdomen for 24 h. The main outcome measures were the plasma concentrations of unconjugated and total rotigotine and its desalkyl metabolites and derived pharmacokinetic parameters (area under the concentration–time curve from time zero to last quantifiable concentration [AUClast], maximum plasma concentration [C max], and body weight- and dose-normalized values).ResultsThe pharmacokinetic analysis included 48 subjects (24 Japanese, 24 Caucasian). The mean apparent dose of rotigotine was 2.0 ± 0.5 mg for Japanese subjects and 2.08 ± 0.58 mg for Caucasians. Plasma concentration–time profiles of unconjugated rotigotine and of the main metabolites were similar for both ethnic groups. Parameters of model-independent pharmacokinetics, C max, time to C max (t max), and AUClast, for unconjugated rotigotine showed no statistically significant differences between Japanese and Caucasian subjects. Values of concentration-dependent pharmacokinetic parameters were higher in female subjects; this difference was minimized after correction for body weight. A statistically significant difference between ethnic groups was observed for total rotigotine concentrations (total rotigotine = unconjugated rotigotine + conjugated rotigotine), with slightly lower values in Caucasians after correction for body weight and apparent dose. No relevant differences were observed between males and females. Inter-individual variability was high. The terminal half-life for unconjugated rotigotine was 5.3 h in Japanese subjects and 5.7 h in Caucasians; corresponding values for total rotigotine were 8.6 h and 9.6 h. Less than 0.1 % of the apparent dose was renally excreted as the parent compound. Renal elimination of total rotigotine covers 11.7 % of absorbed dose in Japanese subjects and 10.8 % of the absorbed dose in Caucasians, whereas the renal elimination via total despropyl rotigotine was 8.2 and 7.1 %, respectively. The corresponding values for total desthienylethyl rotigotine were 3.5 % in Japanese subjects and 4.2 % Caucasians. Most adverse events were mild in intensity and typical for dopamine agonists or for transdermal therapeutics.ConclusionAdministration of a single patch delivering 2 mg/24 h rotigotine resulted in comparable pharmacokinetic profiles in Japanese and Caucasian subjects. The rotigotine transdermal patch was generally well-tolerated. Our findings suggest similar dose requirements for Japanese and Caucasian populations.

Real-Time Monitoring of Chemical Transmission in Slices of the Murine Adrenal Gland

The real-time electrochemical detection of catecholamine secretion from murine adrenal slices using fast-scan cyclic voltammetry (FSCV) and amperometry at carbon fiber microelectrodes is described. Bright-field and immunofluorescent microscopy supported that chromaffin cells in the adrenal medulla are organized into clusters and positively stain for tyrosine hydroxylase confirming that they are catecholaminergic. Spontaneous exocytotic catecholamine events were observed inside chromaffin cell clusters with both FSCV and amperometry and were modulated by the nicotinic acetylcholine receptor antagonist hexamethonium and low extracellular calcium. Reintroduction of extracellular calcium and pressure ejection of acetylcholine caused the frequency of spikes to increase back to predrug levels. Electrical stimulation caused the synchronous secretion from multiple cells within the gland, which were modulated by nicotinic acetylcholine receptors but not muscarinic receptors or gap junctions. Furthermore, electrically stimulated release was abolished with perfusion of low extracellular calcium or tetrodotoxin, indicating that the release requires electrical excitability. An extended waveform was used to study the spontaneous and stimulated release events to determine their chemical content by FSCV. Consistent with total content analysis and immunohistochemical studies, about two thirds of the cells studied spontaneously secreted epinephrine, whereas one third secreted norepinephrine. Whereas adrenergic sites contained mostly epinephrine during electrical stimulation, noradrenergic sites contained a mixture of the catecholamines showing the heterogeneity of the adrenal medulla.

Enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, potential adrenergic ligands, by analytical and preparative liquid chromatography on amylose chiral stationary phases and characterization of the enantiomers

To obtain milligram amounts of the enantiomers of benzoxazolinone derivatives to be tested for binding to adrenergic sites, analytical HPLC methods using derivatized amylose chiral stationary phases were developed for the direct enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, derivatives with one or two chirals centers. The separations were made using normal phase methodology with a mobile phase of n-hexane-alcohol (ethanol, 1-propanol, or 2-propanol) in various proportions, and silica-based amylose (tris-3, 5-dimethylphenylcarbamate) Chiralpak AD and (tris-(S)-1-phenylethylcarbamate) Chiralpak AS columns. The effects of concentration of various aliphatic alcohols in the mobile phase were studied. The best separation was achieved on Chiralpak AS, so preparative HPLC was set up with this chiral stationary phase using a mobile phase consisting of n-hexane-alcohol using isocratic conditions and multiple repetitive injections. Physicochemicals properties of enantiomers were reported The effect of structural features of the solutes on discrimination between the enantiomers was examined. Limit of detection (LD) and limit of quantification (LQ) were determined using both ultra-violet (UV) and evaporative light-scattering detection (ELSD).

Comparative Chromatographic Analysis and Pharmacodynamic Activities of Garcinia Kola Nut Extracts

Objectives: To compare the pharmacodynamic activities of Garcinia Kola nut (GK) extract with known antiglaucoma medication using the movements and sites of separation on thin layer chromatography. Study Design: Experimental Study Materials and Methods: Garcinia kolanuts were obtained from Ogun state. Drugs in comparison were 0.5% Betaxolol, 0.5% Timotol maleate and 2% Pilocarpine. Extraction of Garcinia Kolanuts was carried out at the Pharmaceutical chemistry laboratory of the CMUL. The antiglaucoma drugs were basified in ammonium hydroxide to extract the active ingredients. The other half of water extract served as control. Comparative Thin layer Chromatography (TLC) studies were done both at the College of Medicine Lagos and Olabisi Onabanjo University, Ago-Iwoye Ogun State, using the 3 known antiglaucoma drugs. Garcinia kolanuts were also sent to Alcon Laboratories in Texas, USA for analysis of its active ingredients. Main Outcome Measure: Extent of movement and corresponding spots at sites of separation of all the antiglaucoma drugs and GK extracts on TLC. Results: Thin layer Chromatography (TLC) showed comparative corresponding separating spots of the extracts with those of the antiglaucoma drugs, and the same extent of movements. The laboratory analysis of the Garcinia Kolanuts extracts showed receptors inhibition of Beta adrenergic sites, adenosine, melatonin, opiate, purine, calcium channel L-type and 5HT5A. Conclusion: Garcinia kolanut extract exhibits a combined effects of all the known antiglaucoma drugs used in comparison. NQJHM Vol. 15 (1) 2005: pp. 30-33

Dopamine binds to α 2-adrenergic receptors in the song control system of zebra finches ( Taeniopygia guttata)

A commonly held view is that dopamine exerts its effects via binding to D1- and D2-dopaminergic receptors. However, recent data have emerged supporting the existence of a direct interaction of dopamine with adrenergic but this interaction has been poorly investigated. In this study, the pharmacological basis of possible in vivo interactions between dopamine and α 2-adrenergic receptors was investigated in zebra finches. A binding competition study showed that dopamine displaces the binding of the α 2-adrenergic ligand, [ 3H]RX821002, in the brain. The affinity of dopamine for the adrenergic sites does not differ between the sexes and is 10- to 28-fold lower than that for norepinephrine. To assess the anatomical distribution of this interaction, binding competitions were performed on brain slices incubated in 5 nM [ 3H]RX821002 in the absence of any competitor or in the presence of norepinephrine [0.1 μM] or dopamine [1 μM]. Both norepinephrine and dopamine displaced the binding of the radioligand though to a different extent in most of the regions studied (e.g., area X, the lateral part of the magnocellular nucleus of anterior nidopallium, HVC, arcopallium dorsale, ventral tegmental area and substantia grisea centralis) but not in the robust nucleus of the arcopallium. Together these data provide evidence for a direct interaction between dopamine and adrenergic receptors in songbird brains albeit with regional variation.

Atypical [(3)H]clonidine binding sites in human caudate and platelets on cryostat-cut sections.

Pharmacological characterization is described for a human imidazoline binding site (I-site) labeled by [(3)H]clonidine using standard autoradiographic method. Under conditions that mask alpha(2)-adrenergic sites, only a single high affinity site was observed in human caudate and blood platelet sections. Affinity constants (K(i)) were highly correlated between the two tissues (r = 0.90, P = 0.0003). This site is dissimilar to classical I(1) and I(2) sites, even though both tissues possess abundant I(1) and I(2) sites by filtration binding methods. It is suggested that the isotonic buffer conditions inherent to the procedure alter drug affinities to the classical I(1) site.

Identification and Characterization of the Imidazoline I2b-binding Sites in the Hamster Brown Adipose Tissue as a Study Model for Imidazoline Receptors

The imidazoline-type compound, MPV-1743, has been found to activate nonshivering thermogenesis (NST) in brown adipose tissue (BAT) of the genetically obese Zucker rats. The regulation of NST in BAT is linked to the catecholamine metabolism, and the imidazoline I 2 -binding sites have been found on the monoamine oxidase, a catecholamine metabolising enzyme. In this study, the I 2 -binding sites of hamster BAT have been characterised using a receptor binding assay with 3 H-idazoxan as a radioligand, and the interaction of MPV-1743 with these I 2 -binding sites has been studied using the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089. Cirazoline was used to determine the specific binding of 3 H-idazoxan to the imidazoline I 2 -binding sites. Rauwolscine was added in the 3 H-idazoxan binding assay in order to inhibit any binding to potential a 2 -adrenergic sites. In the presence of rauwolscine mask 3 H-Idazoxan labelled a population of non-adrenergic binding sites expressing the properties of the imidazoline I 2b -receptor subtype similar to that found in the rat liver (cirazoline » guanabenz = amiloride » clonidine). The binding of 3 H-idazoxan to the I 2b -binding sites could be displaced by the imidazole compounds with the following affinities: detomidine (K iHigh 9.2nM; K iLow 3200nM), MPV-2088 (K iHigh 19nM; K iLow 760nM) and MPV-2089 (K iHigh 190nM; K iLow 1300 nM), atipamezole (3500 nM) and dexmedetomidine (K i 8400 nM). These results have shown that the hamster BAT contains the imidazoline I 2b -binding sites with heterogeneous binding properties for some test compounds. In addition, the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089, had high affinity to these BAT imidazoline I 2b -binding sites. Therefore, it is suggested that the regulation of NST in the hamster BAT may be an attractive model to study the role of imidazoline I 2b -binding sites.

Effects of BP 2-94, a selective H 3-receptor agonist, on blood flow and vascular permeability of the rat mandibular incisor pulp

Pulpal blood-flow changes were monitored by laser Doppler flowmetry after electrical stimulation of the mandibular incisor. Stimuli of 10 s (50 μA, 2 ms, 20 Hz) were applied to the incisors of untreated animals and longer stimulations (5 min) were applied in animals treated with the α-blocker phenoxybenzamine. Changes in vascular permeability in the dental pulp were measured by Evans blue extravasation following resection of the superior cervical ganglion. In these groups, a selective agonist of H 3 receptors, BP2-94 (1.5 and 15 mg/kg), and an H 3-antagonist ciproxifan (1 mg/kg) were administered. The effects of these drugs were compared with those obtained from animals treated only with the vehicle (methylcellulose 1%). Basal pulpal blood-flow was not affected significantly by BP2-94 or ciproxifan. The vasoconstriction induced in the group of intact rats by electrical stimulation of 10 s is decreased in amplitude and duration at the higher dose of BP2-94 by 58 and 40%, respectively ( P<0.05, n=5). In the sympathectomized animals, plasma extravasation was significantly increased at 15 mg/kg BP2-94 (+100%, P<0.01, n=5). These results suggest that H 3 receptors may participate in the regulation of changes in vessel contraction and permeability provoked by electrical stimulation of the dental pulp. However, the non-selective effects of the H 3 agonists reacting on adrenergic sites and H 1 receptors could explain a part of the results.

Carotid body I 1-imidazoline receptors: Binding, visualization and modulatory function

The carotid body is influenced by many neurotransmitter receptors. A novel receptor specific for imidazolines has been implicated in cardiorespiratory regulation in the brain. To test for both I 1-imidazoline and α 2-adrenergic receptors, which also recognize imidazolines, specific [ 125I] p-iodoclonidine binding to carotid body membranes was characterized. The specific α 2-agents epinephrine (100 μM) or SK&F 86466 (10 μM) inhibited only a portion of specific [ 125I] p-iodoclonidine binding in both cat and rabbit carotid bodies, indicating the presence of I 1-imidazoline as well as α 2-adrenergic sites. The distribution of [ 125I] p-iodoclonidine binding sites was visualized autoradiographically. The cat carotid body was intensely labeled by [ 125I] p-iodoclonidine, with both I 1-imidazoline and α 2-adrenergic sites expressed. The relevance of I 1-imidazoline receptors in modulation of chemosensory discharge was determined in seven cats after α 2-adrenergic blockade. Clonidine (100 μg/kg) facilitated chemosensory activity particularly under hypoxia. We conclude that I 1-imidazoline receptors are expressed within the carotid body and may potentiate chemosensory discharge, in contrast to the inhibitory action of α 2-adrenergic receptors.

Adrenergic Receptors in Alzheimer’s Disease Brain: Selective Increases in the Cerebella of Aggressive Patients

In this study, the distribution and concentration of beta1, beta2, and alpha2 adrenergic receptors were examined in the frontal cortex, hypothalamus, and cerebellum of Alzheimer's disease (AD) and age-matched control human brains by receptor autoradiography. The purpose of this study was to detect changes in adrenergic receptor concentrations in key areas of the brain known to affect behavior. For these studies, [125I]iodopindolol ([125I]IPIN) was used to visualize total beta adrenergic sites (with ICI-89,406 and ICI-118, 551 as subtype-selective antagonists to visualize beta2 and beta1 receptors, respectively). [3H]UK-14,304 was used to localize the alpha2 sites. Essentially no significant difference in adrenergic receptor concentration was found between total AD cases taken together and control patients. It was found, however, that there were important distinctions within the AD group when cases were subdivided according to the presence or absence of aggression, agitation, and disruptive behavior. Aggressive AD patients had markedly increased (by approximately 70%) concentrations of alpha2 receptors in the cerebellar cortex compared with nonaggressive patients with similar levels of cognitive deficit. The levels of cerebellar alpha2 receptors in aggressive AD patients were slightly above the healthy elderly controls, suggesting that these receptors are preserved and perhaps increased in this subgroup of AD. beta1 And beta2 adrenergic receptors of the cerebellar cortex showed smaller but significant ( approximately 25%) increases in concentration in aggressive AD subjects versus both nonaggressive AD patients and controls. No significant differences were found in adrenergic receptor concentrations within the frontal cortex or hypothalamus. These results point out the importance of distinguishing behavioral subgroups of AD when looking for specific neurochemical changes. These autoradiographic results may reflect the importance of the cerebellum in behavioral control.

Design and stereoselective synthesis of conformationally constrained analogues of Zimeldine

Most of the commonly used antidepressant drug (e. g. imipramine and amitriptyline) have cardiovascular side effects (orthostatic hypotension, tachycardia, arrhythmia) due to the action of these drugs at cholinergic and adrenergic sites (Ankier, 1986). The goal of this research program is to design and sythesize very selective inhibitors for the uptake of either, but not both, norepinephrine (NE) or serotonin (5HT). Examining the relation between the conformation of these molecules and their selectivity for inhibition of the uptake of NE or 5HT could provide an understanding of the molecular mechanisms involved. Such information could lead to even safer antidepressant drugs with diminished cardiovascular side effects. Furthermore, drugs that are selective for inhibition of the uptake of 5HT (e.g. fluoxetine) show promise as aspects for obesity control (Grunewald et al., 1986a). In order to find new drugs with enhanced selectivity for the uptake of 5HT which could be useful as antidepressants requires an understanding of the structural features of these drugs that results in their selectivity for the 5HT uptake carrier. As part of a search for the explanation of the dramatic pharmacological difference between (Z)-Zimeldine (1, a selective inhibitor of the reuptake of serotonin) and (E)-Zimeldine (2, a selective inhibitor of norepinephrine) in presynaptic nerve endings (Hoberg et al., 1981), we sought a convenient synthesis of conformationally defined analogues of 1 and 2 in the quinoline and naphthalene skeleton. We wondered if subtle changes in low energy conformations of the

Effect of I1-imidazoline receptor activation on responses of hypoglossal and phrenic nerve to chemical stimulation.

Sedation elicited by some centrally acting antihypertensive agents may interfere with respiratory control, and by selectively inhibiting upper airway dilating muscle activity it may facilitate obstructive sleep apnea. Autoradiographic studies with [125I]p-iodoclonidine in the presence of 10 microM epinephrine to block alpha 2-adrenergic sites or 100 nM moxonidine to mask I1-imidazoline sites show that both I1- as well as alpha 2-sites are localized in putative chemosensory areas of the rostral ventrolateral medulla in the cat. We sought to determine the effect of activating I1 and alpha 2-receptors on central chemosensitivity by using moxonidine as a selective I1 agonist, clonidine as a mixed I1/alpha 2 agonist, SK&F-86466 as a specific alpha 2-antagonist, and efaroxan as a mixed I1/alpha 2 antagonist. We recorded responses of phrenic, hypoglossal, and cervical sympathetic nerve activities to progressive hypercapnia after hyperventilation to apnea. Moxonidine (3-100 micrograms/kg i.v.) caused dose-dependent decreases in tonic cervical sympathetic nerve activity and blood pressure, but had no effect on the CO2 threshold (after 30 or 100 micrograms/kg moxonidine, phrenic nerve activity reappeared at 5.8 +/- 0.2% CO2 versus 5.6 +/- 0.3% CO2 in control). Following moxonidine, the slope of the steep portion of the CO2 response tended to increase (10.3 +/- 1.8 versus 7.3 +/- 0.9). Peak phrenic nerve activity was comparable to control at 7.5% CO2 (20 +/- 2 U in control) and at 9.5% CO2 (30 +/- 3 versus 27. +/- 2 U). Similarly, the response of hypoglossal and inspiratory phasic cervical sympathetic nerve activity to a progressive CO2 rise was not affected by moxonidine. By contrast, clonidine in the same doses decreased CO2 sensitivity, because the CO2 threshold was elevated from 5.3 +/- 0.5% to 6.7 +/- 0.4% (p < 0.001). The slope of the CO2 response was decreased from 9.7 +/- 1.9 to 7.4 +/- 1.3 (p = 0.05). Peak phrenic nerve activity was reduced at 7.5% CO2 (11 +/- 5 versus 25 +/- 2 U; p < 0.05) and at 9.5% CO2 (21 +/- 4 versus 33 +/- 2 U; p = 0.06). Clonidine selectively inhibited the response of hypoglossal nerve activity to CO2. The depressive effects of clonidine were reversed by alpha 2-blockade with SK&F-86466 (0.5 or 1 mg/kg). Inspiratory phasic cervical sympathetic nerve activity increased after SK&F-86466 in parallel with phrenic and hypoglossal nerve activity, but the tonic component of cervical sympathetic nerve activity and blood pressure increased only transiently.(ABSTRACT TRUNCATED AT 400 WORDS)

Unique profiles of the alpha 1-, alpha 2-, and beta-adrenergic receptors in the developing cortical plate and transient embryonic zones of the rhesus monkey

Film receptor autoradiography was used to study the distribution of alpha 1-, alpha 2-, and beta-adrenergic receptors in the occipital lobe of developing rhesus monkeys ranging in age from the 65th embryonic day (E65) to adulthood (5 years of age). The study shows that each adrenergic receptor subtype has a unique distribution in the cortical plate and transient embryonic zones of the developing cerebral wall. For example, alpha 1 receptors are prominent throughout the proliferative ventricular and subventricular zones while alpha 2 receptors in the same zones form three distinct bands interdigitated with three bands rich in beta sites. There are also considerable temporal differences in the emergence of adrenergic receptor subtypes in specific embryonic zones. For instance, the high density of alpha 1 sites in the germinal zones occurs only in conjunction with intensive proliferative activity. In contrast, beta receptors emerge in these zones after the majority of cortical neurons have been generated. The transient embryonic zones often display higher densities of adrenergic sites than the cortical plate itself. In particular, the subplate zone subjacent to the developing visual cortex contains the highest density of alpha 2-adrenergic sites in the cerebral wall throughout all prenatal ages studies. Finally, the regional differences in the density of beta-adrenergic receptors are evident in the subplate zone underlying the prospective striate and extrastriate cortex before such differences emerge in the cortical plate. The early appearance, unique pattern of distribution, and time-dependent changes of adrenergic receptors in the transient embryonic zones suggest their involvement in the regulation of the cortical development.

A novel mechanism of action for hypertension control: Moxonidine as a selective I1-imidazoline agonist

Sympathoadrenal inhibition by a direct action within the central nervous system is an advantageous route to blood pressure control. Stimulation of brain alpha 2-adrenergic receptors is one mechanism for sympathoadrenal suppression, but comes at the cost of nonspecific depression of CNS function, including sedation and decreased salivary flow. Evidence is accumulating for a second pathway for pharmacological control of sympathoadrenal outflow, mediated by a novel receptor specific for imidazolines. First-generation central antihypertensive agents, which are imidazolines such as clonidine, act primarily to stimulate these I1-imidazoline receptors in the rostral ventrolateral medulla oblongata (RVLM) to lower blood pressure, but have sufficient agonism at alpha 2-adrenergic receptors to produce side effects. Second-generation centrally acting antihypertensive agents, such as moxonidine and rilmenidine, are selective for I1 relative to alpha 2 receptors. The reduced alpha 2 potency of these agents correlates with reduced severity of side effects. In this study we further established the selectivity of moxonidine for I1-imidazoline sites by characterizing the direct interaction of [3H]moxonidine with these receptors in the RVLM and in adrenomedullary chromaffin cells. [3H]Moxonidine preferentially labeled I1-imidazoline sites relative to alpha 2-adrenergic sites, only a small portion of which were labeled in the RVLM. [3H]Moxonidine binding to I1-imidazoline sites was modulated by guanine nucleotides, implying that I1-imidazoline sites may be membrane receptors coupled to guanine nucleotide binding regulatory proteins (G proteins). Receptor autoradiography with [125I]p-iodoclonidine confirmed the presence of I1-imidazoline sites in the RVLM and other areas of the brainstem reticular formation. In contrast, alpha 2-adrenergic sites were mainly localized to the nucleus of the solitary tract. Moxonidine selectively displaced [125I]p-iodoclonidine binding from reticular areas, including the RVLM. In vivo studies in SHR rats confirmed the ability of moxonidine to normalize hypertension by an action within the RVLM and confirmed the correspondence of I1 binding affinity and antihypertensive efficacy. We also discuss prior literature on the cardiovascular pharmacology of imidazolines, reinterpreting previous studies that only considered alpha-adrenergic mechanisms.

Expression of non-adrenergic imidazoline sites in rat cerebral cortical astrocytes.

Clonidine and related imidazoline agents, beside binding to alpha 2-adrenergic receptors, have been shown to bind to a non-adrenergic site (imidazoline sites) in brain and peripheral tissues. However, which cell types in brain, namely neurons or glia, express this binding site and the cellular effects of activation of this site are not known. We investigated the cellular localization of imidazoline binding sites in cultured rat cortical astrocytes and neurons. Membranes prepared from cultured astrocytes showed specific, high affinity binding (KD: 4 nM) for 3H-idazoxan with about tenfold higher number of binding sites than alpha 2-adrenergic sites (Bmax: 220 vs. 20 fmol/mg protein). Displacement studies exhibited the rank order of potency: cirazoline > idazoxan > amiloride > clonidine >>> epinephrine = ruawolscine defining this site as I-2a subtype of imidazoline binding sites. Moreover, the binding was inhibited by K+ but not by Na+, another characteristic of imidazoline binding sites. In contrast, membranes prepared from cultured neurons showed fewer binding sites for 3H-idazoxan that were completely displayed by adrenergic agents. Incubation of astrocytes with idazoxan, but not rauwolscine, resulted in a concentration-dependent increase in the levels of mRNA for the astrocyte specific molecule glial fibrillary acidic protein. We conclude that (a) the non-adrenergic imidazoline binding sites are expressed in astrocytes but not in neurons in rat cerebral cortex and (b) these "receptors" may influence astrocyte physiology by regulating the levels of GFAP.

Phenylethanolaminotetralines compete with [ 3H]dihydroalprenolol binding to rat colon membranes without evidencing atypical β-adrenergic sites

[ 3H]Dihydroalprenolol ([ 3H]DHA) specific binding (determined by the difference in the presence and absence of 20 μM (−)isoprenaline) to rat colon membranes was saturable ( B max = 39.6 frnol/ mg protein), of high affinity ( K d = 0.87nM) and stereospecific ( ic 50 330 and 3510 nM for (-)- and (+)isoprenaline, respectively); the Hill coefficient was close to one, indicating binding homogeneity. [ 3H]DHA (0.6 nM) specific binding was potently inhibited ( K i range 1.9–3.3 nM) by the non-selective β-adrenoceptor antagonists pindolol, alprenolol and propranolol, but not by the non-adrenergic compounds 5-hydroxytryptamine, 8-hydroxydipropylaminotetraline, methysergide, dopamine and verapamil ( K i > 10,000 nM). The selective β 1- and β 2-adrenoceptor antagonists CGP 20,712A and ICI 118,551 resulted in biphasic competition binding curves, whose low and high affinity components were compatible with two populations of binding sites accounting for about 75 (β 2) and 25% (β 1) of total sites. The relative competing potencies of reference adrenergic agonists also suggested a prevalence of β 2-adrenergic sites. The new agonists phenylethanolaminotetralines (PEATs), highly selective for the atypical β-adrenoceptors whose abundance in rat colon has been confirmed by comprehensive functional studies, had variable affinity for the [ 3H]DHA-labelled sites depending on chirality, but with no substantial correlation with their pharmacological potency. Only 40% of [ 3H]DHA binding, at a concentration about 10 times its K d for high affinity sites (β 1 and β 2), was prevented by saturating concentrations of isoprenaline. Under this condition, the representative PEAT, SR 58611A, highly potent and selective for atypical β-adrenoceptors in functional tests, and its pharmacologically inactive enantiomer, both inhibited the residual binding equipotently. In conclusion, [ 3H]DHA binding did not detect atypical β-adrenoceptor sites in rat colon membranes, most probably because of its weaker affinity for them than for the coexisting β 1 and β 2 sites. PEAT stereoisomers proved essential for assessing both the stereospecificity and the functional significance of this atypical binding and to compare their affinity for [ 3H]DHA-labelled sites and pharmacological potency.

Adrenergic and nonadrenergic effects of imidazoline and related antihypertensive drugs in the brain and periphery.

In radioligand binding studies the alpha 2-adrenoceptor antagonist ligand [3H]yohimbine binds exclusively to adrenergic sites. However, in addition to binding to alpha 2-adrenoceptor sites, two other ligands, [3H]clonidine and [3H]idazoxan, also bind at nonadrenergic imidazoline sites. Many of the compounds with a high affinity for these imidazoline sites are centrally acting antihypertensive drugs and there is some evidence that these sites are involved in blood pressure regulation. With chronic treatment of rabbits with guanabenz, clonidine, and rilmenidine, down regulation of alpha 2 but not imidazoline sites occurred in forebrain and hindbrain with guanabenz treatment, and in hindbrain with clonidine treatment. However, no change in either imidazoline or alpha 2-adrenoceptor binding site number occurred in animals given chronic rilmenidine treatment. This rank order of effect, guanabenz greater than clonidine greater than rilmenidine, is consistent with the alpha 2-adrenoceptor activation by the three drugs in the periphery. In contrast, chronic treatment with guanabenz, clonidine, and rilmenidine had similar effects on blood pressure, heart rate, and responses to intracisternal clonidine. We suggest that stimulation of alpha 2-adrenoceptors cannot account for all the changes in cardiovascular responses observed on chronic treatment and that activation of nonadrenergic imidazoline-preferring sites may contribute to the antihypertensive properties of imidazoline and related compounds.

Comparative evaluation of lisuride and terguride, ergot alkaloid derivatives, on the field-stimulated vas deferens of mouse

The ergot alkaloid derivatives, lisuride (LIS) and terguride (TDHL), known to interact with central dopamine receptors as agonist and partial agonist, respectively, were studied on the field-stimulated mouse vas deferens, where recently the existence of presynaptic dopamine receptors has been evidenced. LIS was a competitive antagonist at prejunctional alpha 2 and DA1 receptors situated on the sympathetic nerve terminals of the mouse, with a pA2 value of 9.2 and 9.1, respectively. LIS was also able to antagonize the effects of LY 171555, selective DA2 agonist, but the type of interaction cannot be conceptualized in terms of competitive antagonism. Likewise, the type of interaction of TDHL with dopaminergic and adrenergic agonist-activated sites is not suggestive of a competitive antagonism. Based on these results, it seems that central and peripheral pharmacologic profiles of LIS and TDHL cannot be overlapped, LIS being a potent DA1- and alpha 2-antagonist with a high degree of specifity for these receptors.

Imidazole binding sites in rabbit kidney and forebrain membranes

1. The binding of [3H]-clonidine, [3H]-idazoxan and [3H]-yohimbine to rabbit forebrain and kidney membranes was compared. 2. Yohimbine bound exclusively to adrenergic sites, idazoxan to non-adrenergic sites and clonidine to both non-adrenergic and adrenergic sites. 3. Differences were observed between the ligands not only in binding at adrenergic and non-adrenergic sites but also between the non-adrenergic binding of [3H]-clonidine and [3H]-idazoxan. 4. However, no tissue specific differences were found.

Rilmenidine lowers arterial pressure via imidazole receptors in brainstem C1 area

We sought to determine the site of action and receptor type responsible for the antihypertensive actions of rilmenidine. an oxazoline analogue of clonidine. In anesthetized paralyzed rats decerebration did not alter the dose dependent reductions in arterial pressure and heart rate elicited by i.v. drug. Rilmenidine microinjected bilaterally into the C1 area of the rostral ventrolateral medulla (RVL), but not nucleus tractus solitarii (NTS) nor caudal ventrolateral medulla (CVL), elicited dose-dependent falls in arterial pressure and heart rate at doses an order of magnitude less than required systemically. Prior microinjection into the Cl area of the selective α 2-adrenoceptor antagonist SKF-86466, even at high doses, failed to modify the hypotension to i.v. rilmenidine. However, microinjection of 3- to 10-fold lower doses of idazoxan, a ligand for imidazole as well as α 2-adrenoceptors, blocked the effects. Rilmenidine also competed with the clonidine analogue [ 3H]p-aminoclonidine ([ 3H]pac) at specific binding sites in membranes of bovine ventrolateral medulla and frontal cortex. In RVL rilmenidine competed with binding to imidazole and α 2-adrenergic binding sites with a 30-fold selectivity for the imidazole binding sites. In frontal cortex binding was of lower affinity and restricted to α 2-adrenergic sites. We conclude that rilmenidine, like clonidine, acts to lower arterial pressure by an action on imidazole receptors in the Cl area of RVL. The higher selectivity of rilmenidine for imidazole to α 2-adrenoceptors as compared to clonidine may explain the lower sedative effects of rilmenidine. Rilmenidine; Oxazoline; α 2-Adrenoceptors; Imidazole receptors; Medulla (rostral ventrolateral); Arterial pressure