Sodium modulation of ³H‐agonist and ³H‐antagonist binding to α₂‐adrenoceptor subtypes

Abstract

1 The a2-adrenoceptors on human platelets and neonatal rat lung were characterized with the agonist and antagonist ligands [3H]-adrenaline and [3H]-RS-15385-197 respectively. A correlation of affinities for 3H-antagonist binding showed the receptors to be of the M2A-(platelet) and x25-(neonatal rat lung) adrenoceptor subtypes, whereas a correlation of affinities for 3H-agonist binding showed the receptors to have similar characteristics (r = 0.88). 2 NaCI (100 mM) had no effect on [3H]- RS-15385-197 13 fold. The affinity of the catecholamines, adrenaline and noradrenaline was significantly decreased, whereas the imidazolines, oxymetazoline and UK-14,304 were much less affected. The affinity of prazosin and imiloxan for [3H]-adrenaline binding was significantly increased in the presence of 10 and 100 mM NaCl. Conversely, the affinity of adrenaline and noradrenaline was decreased in the presence of NaCl, and there was no change in the affinity of the imidazoline agonists. 4 In the human platelet, NaCl had no effect on the affinity of prazosin for [3H]-RS-15385-197 binding but the affinity of imiloxan was significantly increased. NaCl significantly decreased the affinity of the catecholamines adrenaline and noradrenaline, whereas the affinity of 304 and oxymatazoline was much less affected. Competition experiments with [3H]-adrenaline in the presence of NaCl in platelets were difficult to characterize as there was no specific binding under these conditions. 5 The results show that both the M2A-and a2B-adrenoceptor subtypes are allosterically regulated by Na+, but only the a2B-subtype showed a significant increase in density. Interestingly, there is a differential regulation of imidazoline (unchanged) and catecholamine (decreased affinity) agonist interactions with these subtypes. Na+ may therefore critically affect receptor subtype selectivity of drugs. The implications for receptor subclassification are discussed.

Comparison of the interaction of agmatine and crude methanolic extracts of bovine lung and brain with α2‐adrenoceptor binding sites

“…The working hypothesis underpinning the present study is that radioligand binding assays, involving the use of a 3H-agonist and a 3H-antagonist, can predict the pharmacological properties of recognized agonists and antagonists (Wilson et al, 1991;MacKinnon et al, 1993). For this to be possible, it is necessary to establish that the radioligands employed identify a pharmacologically homogeneous population of sites.…”

“…For example, x2-adrenoceptor agonists possess a higher affinity for a2A/D-sites labelled by 3H-agonists (e.g., [3H]-adrenaline or [3HI-UK-14304) compared to the same sites labelled by 3H-antagonists (e.g., [3H]-rauwolscine). Conversely, antagonists appear to possess a higher affinity for a2-adrenoceptors labelled by 3Hantagonists compared to the corresponding site labelled by an 3H-agonist (Gleason & Hieble, 1991;MacKinnon et al, 1993; see also Wilson et al, 1991). Examples of preparations possessing x2-adrenoceptors that exhibit this type of behaviour include human platelets (Garcia-Seville & Fuster, 1986;MacKinnon et al, 1993).…”

“…Conversely, antagonists appear to possess a higher affinity for a2-adrenoceptors labelled by 3Hantagonists compared to the corresponding site labelled by an 3H-agonist (Gleason & Hieble, 1991;MacKinnon et al, 1993; see also Wilson et al, 1991). Examples of preparations possessing x2-adrenoceptors that exhibit this type of behaviour include human platelets (Garcia-Seville & Fuster, 1986;MacKinnon et al, 1993). HT-29 cells (Turner et al, 1985;Bylund et al, 1988;Gleason & Hieble, 1991) and rat cerebral cortex membranes (Wallace et al, 1994): preparations that belong to the C2A/D subtypes.…”

Subtype determination of presynaptic ?2-autoreceptors in the rabbit pulmonary artery and human saphenous vein

“…Table 4 Ratios of inhibition constants (Ki) of the a-adrenoceptor ligands phentolamine and rauwolscine, determined in radioligand binding experiments, and ratios of potencies of the ligands at the a2-autoreceptors in rabbit pulmonary artery and human saphenous vein. In the case of the radioligand binding experiments, means_+95% confidence interval of the ratios of the Ki values in the tissues or cells indicated in the footnotes were calculated Phentolamine/rauwolscine a2a-sites a 25 -+9 a2B-sites b 7.3 +5.9 azc-sites c 178 -+95 a2i)-sites a 0.30-+0.20 Rabbit pulmonary artery e 33 Human saphenous vein e 30 a Human platelets (Bylund et al 1988 a); chicken pineal gland (Bylund et al 1988b); human spinal cord (Lawhead et al 1992); COS-C10 b Neonatal rat lung (Bylund et al 1988a, Bylnnd et al 1992, MacKinnon et al 1993); NG108-15 cells (Bylund et al 1988a); rat kidney (Michel et al 1989); human a2C2 clone (Marjam~i et al 1993) c Opossum kidney cells (Murphy and Bylund 1988, Blaxall et al 1991; opossum kidney (Blaxall et al 1991); human a2C4 clone (Regan et al 1988, Marjamfiki et al 1993); COSa2C4 d Rat submaxillary gland (Michel et al 1989, Renouard et al 1994 In agreement with the pharmacological properties of a2A, a2B, and a2c-adrenoceptor subtypes outlined in the Introduction, the possibility that the a2-adrenoceptors in the rabbit pulmonary artery belong to the a2B-or a2c-subtype can be excluded (1) by the low absolute potency of prazosin and (2) by the high relative potency of oxymetazoline compared to prazosin. (1) Prazosin up to 1 gmol/l failed to substantially increase noradrenaline release (present study; Starke and Docherty 1980;G6thert et al 1983;Nedergaard 1988) and at 1 gmol/1 it did not counteract the clonidine-and rilmenidine-induced inhibition of evoked noradrenaline release (Verbeuren et al 1986).…”

Autoradiographic Demonstration of Increased α2‐Adrenoceptor Agonist Binding Sites in the Hippocampus and Frontal Cortex of Depressed Suicide Victims

“…The a2 -adrenoceptor autoradiographic labeling was performed as previously described (Pazos et al ., 1988 ;Pascual et al ., 1992). After a 15-min preincubation at room temperature in Tris-HCI buffer (50 mM, pH 7.7) containing 0.1 mM MnC1 2 , tissue sections were incubated with 6 nM ['H]UK 14304 (an imidazoline drug that is a mixed a2A/s/c-adrenoceptor agonist; Uhl6n et al ., 1992 ;Mackinnon et al ., 1993) for 90 min in the same buffer, also at room temperature. After the incubation, sections were washed for 5 min in ice-A.…”