Supersensitivity of beta-adrenoceptor coupled adenylate cyclase in pulmonary tissue of the spontaneously hypertensive rat

Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). However, sensitivity to Gpp[NH]p, isoproterenol plus GTP or Gpp[NH]p was significantly greater in the SHR. Beta-receptor density measured by [3H]DHA binding was unaltered. The dissociation constant, Kd, revealed a significantly greater binding affinity of the radioligand in the SHR (6.23 +/- 0.45 nM) compared with the WKY (8.53 +/- 0.82 nM). Activity of Gs was assessed by complementing S49 cyc- acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp[NH]p, and isoproterenol plus Gpp[NH]p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary Gs in the hypertensive rats.     

Neurotensin receptors on the rat liver plasma membranes

Neurotensin (NT) is now classified as a brain-gut peptide in the central nervous system and gastrointestinal tract. In the present study, we characterized the NT receptors on the rat liver plasma membranes. The specific binding of [3H]NT was time dependent, reversible, and saturable. Scatchard analysis of the specific binding data yielded two classes of binding sites, a high affinity site and a low affinity site. The average maximum number of binding sites (Bmax) amounted to 13.3 +/- 1.1 fmol/mg protein at high affinity site and 122.3 +/- 21.5 fmol/mg protein at low affinity site, respectively. The dissociation constant (Kd) had values of 0.39 +/- 0.01 nM at high affinity site and 8.1 +/- 1.1 nM at low affinity site, respectively. The amount of specifically bound [3H]NT was significantly reduced in the presence of mono and divalent cations, EDTA, EGTA and a peptidase inhibitor bacitracin, NT1-13 competed with [3H]NT for its binding site with an IC50 of 0.19 nM at high affinity site (0.2 nM concentration of [3H]NT) and 0.7 nM at low affinity site (4.0 nM concentration of [3H]NT). Xenopsin, a NT analogue separated from the skin of Xenopus laevis, was equipotent (IC50 0.75 nM) with NT1-13 at 4.0 nM concentration of [3H]NT. C-terminal sequence of NT contains the structure necessary for interaction with NT binding sites whereas N-terminal sequence had no binding activity. Since NT has a hyperglysemic and a hypercholesterolemic effects in rats, these NT receptors on the rat liver plasma membranes may be involved in the hyperglycemia and/or hypercholesteroremia induced by NT.     

Altered transverse tubule dihydropyridine receptor binding in malignant hyperthermia

Transverse tubule (TT) membrane vesicles have been isolated from the skeletal muscle of normal and malignant hyperthermia-susceptible (MHS) pigs. MHS and normal TT did not differ in the distribution of the major proteins, cholesterol, or phospholipid content, (Na+ + K+)-ATPase activity, [3H]ouabain binding, Ca2+-ATPase activity, Mg2+-ATPase activity, or [3H]saxitoxin binding. Furthermore, in the presence of micromolar Ca2+, MHS and normal TT did not differ significantly in the KD values for either [3H]nitrendipine binding (2.7 +/- 0.6 and 3.3 +/- 0.5 nM, respectively) or (-)-[3H]desmethoxyverapamil ([3H]D888) binding (7.2 +/- 0.9 and 6.4 +/- 0.6 nM, respectively). However, in contrast to normal TT, MHS TT exhibited a significantly decreased Bmax for both [3H]nitrendipine binding (26.4 +/- 5.4 for MHS versus 40.6 +/- 3.7 pmol/mg protein for normal TT) and [3H]D888 binding (17.8 +/- 7.0 for MHS versus 37.4 +/- 5.9 pmol/mg protein for normal TT). At calcium concentrations greater than 0.1 mM, there was a greater inhibition of [3H]nitrendipine binding to normal than to MHS TT such that binding was now similar for both preparations. As with purified TT, [3H]nitrendipine binding to MHS muscle homogenates was significantly less than to normal muscle homogenates (109 +/- 20 versus 211 +/- 19 fmol/mg protein, for MHS and normal TT, respectively); this difference was not apparent when 100 mM CaCl2 was included in the binding medium. We conclude that the altered MHS TT dihydropyridine receptor properties may reflect an adaptation of the TT voltage sensing mechanism to the abnormal sarcoplasmic reticulum calcium release channel regulation in MHS muscle.     

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.     

(+)-[3H]MK-801 Binding Sites in Postmortem Human Brain

The pharmacological specificity and the regional distribution of the N-methyl-D-aspartate receptor-associated 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) binding sites in human postmortem brain tissue were determined by binding studies using (+)-[3H]MK-801. Scatchard analysis revealed a high-affinity (KD = 0.9 +/- 0.2 nM, Bmax = 499 +/- 33 fmol/mg of protein) and a low-affinity (KD = 3.6 +/- 0.9 nM, Bmax = 194 +/- 44 fmol/mg of protein) binding site. The high-affinity site showed a different regional distribution of receptor density (cortex greater than hippocampus greater than striatum) compared to the low-affinity binding site (cerebellum greater than brainstem). The rank order pharmacological specificity and stereoselectivity of the high-(cortex) and low-(cerebellar) affinity binding sites were identical. However, all compounds tested showed greater potency at the high-affinity site in cortex. The results indicate that (+)-[3H]MK-801 binding in human postmortem brain tissue shows pharmacological and regional specificity.     

19-hydroxyandrostenedione does not modulate [3H]aldosterone binding to human mononuclear leucocytes and rat renal cytosol

To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.     

Interactions of radiolabelled ligands with specific receptors: an analysis

The binding and displacement of beta-adrenoceptor blockers, [3H]propranolol ([3H]PRP) and [3H]dihydroalprenolol ([3H]DHA), were studied on isolated rat erythrocytes, their membranes and ghosts; the binding of [3H]DHA and a M-cholinoceptor blocker, [3H]quinuclidinylbenzylate ([3H]QNB), on cerebral cortex membranes. In all experiments, ligand-receptor interactions conformed to a model of two pools of receptors in the same effector system and the binding of two ligand molecules to the receptor. The results were similar for the displacement of [3H]PRP, [3H]DHA and [3H]QNB with propranolol, dihydroalprenolol and quinuclidinyl-benzylate, respectively. The parameters of [3H]PRP to beta-adrenoceptor binding for intact erythrocytes were: Kd1 = 0.74+/-0.07 nM, Kd2 = 14.40+/-0.41 nM, B1 = 24+/-2 unit/cell, B2 = 263+/-5 unit/cell; for ghosts, Kd1 = 0.70+/-0.17 nM, Kd2 = 19.59+/-2.59 nM, B1 = 9+/-1 fmol/mg protein, B2 = 39+/-4 fmol/mg protein. Receptor affinities were similar in erythrocytes and ghosts; on the ghost membrane, the number of receptors was considerably lower (B1 = 2 unit/cell, B2 = 6 unit/cell). The parameters of [3H]QNB to M-cholinoceptor binding of the cerebral cortex membrane were the following: Kd1 = 0.43 nM, Kd2 = 2.83 nM, B1 = 712 fmol/mg, B2 = 677 fmol/mg.     

Alterations in central and peripheral adrenergic receptors in deoxycorticosterone/salt hypertensive rats

The treatment of uninephrectomized rats with deoxycorticosterone (DOCA) and salt for 6 weeks caused a significant systolic hypertension and cardiac and renal hypertrophy. There was a significant decrease in the density of cardiac α₁- and β-, and renal α₁-adrenoceptors in DOCA/salt hypertensive rats, as compared to uninephrectomized salt loaded control rats. In contrast, the cerebral cortex, corpus striatum, hippocampus and hypothalamus/thalamus of hypertensive rats showed a significant increase in adrenoceptor binding in these hypertensive rats. In contrast, muscarinic cholinergic receptors and [³H]yohimbine binding sites were not altered in most tissues of the hypertensive rats. The present study suggests an important role for central and peripheral α₁- and β-adrenoceptors in the pathogenesis of hypertension.     

Ontogeny of Calcium Antagonist Binding Sites in Chick Brain and Heart

The ontogeny of chick brain and heart ventricle calcium antagonist binding sites was determined, using [3H]nitrendipine ([3H]NDP), as the ligand. The binding of [3H]NDP to adult heart and brain was kinetically very similar, with the former displaying a KD of 0.28 +/- 0.02 nM and a Bmax of 138 +/- 17 fmol/mg protein, and the latter a KD of 0.30 +/- 0.02 nM and a Bmax of 160 +/- 12 fmol/mg protein. The binding site in both brain and heart was highly specific for dihydropyridine calcium antagonists, such as nifedipine, nimodipine, and nisoldipine, since these drugs were several orders of magnitude more potent as inhibitors of [3H]NDP binding than verapamil, methoxyverapamil, or diltiazem. The developmental appearance of [3H]NDP binding sites in brain was rather gradual, with adult levels being attained just prior to birth. This was in contrast to the profile in heart ventricle which showed essentially adult levels at seven days gestation. The acquisition of [3H]NDP binding sites in chick brain roughly paralleled the onset of neuronal maturation and functional activity. In both chick brain and heart, verapamil and methoxyverapamil were weak inhibitors of [3H]NDP binding. However, the inhibition of binding in both tissues was markedly biphasic, with only 50% of the binding sites being susceptible to inhibition by each agent, suggesting that multiple calcium antagonist binding sites may exist in both tissues.     

Lipolysis and beta-adrenergic receptor binding on adipocytes of spontaneously hypertensive rats

Adipocytes from spontaneously hypertensive rats demonstrated a blunted lipolytic response to isoproterenol and dibutyryl cyclic AMP. (-)-[3H]Dihydroalprenolol binding was examined in adipocytes from normotensive and spontaneously hypertensive rats. Increasing concentrations of isoproterenol decreased total (-)-[3H]dihydroalprenolol binding to intact cells from normotensive rats, and the efficacy of competition was decreased in adipocytes from spontaneously hypertensive rats. Scatchard analysis indicated that the number of (-)-[3H]dihydroalprenolol binding sites and the affinity of dihydroalprenolol binding were comparable between normotensive and spontaneously hypertensive rats. Isoproterenol- and Gpp(NH)p-stimulated adenylate cyclase activity was consistently depressed in adipocyte membranes from spontaneously hypertensive rats as compared to normotensive rats. No difference in fluoride-stimulated adenylate cyclase activity was observed. The blunted lipolytic and cyclic AMP response to isoproterenol in these cells suggest a postreceptor lesion of the lipolytic pathway (possibly the guanine nucleotide regulatory protein) in adipocytes from spontaneously hypertensive rats. The blunted lipolytic response to dibutyryl cyclic AMP suggests defective regulation of lipolytic enzymes at the protein kinase-hormone-sensitive lipase level.     

Evaluation of prostaglandin-receptors in human and rat liver: interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50:2.5 +/- 1.7, (6.1 +/- 2.1) microM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9 +/- 0.9 (2.0 +/- 0.8) microM. The Scatchard analysis on [3H]PGE1- as well as on [3H]iloprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3 +/- 5.2 (21.3 +/- 4.3) fmol/mg protein and a Kd of 2.1 +/- 1.8 (1.9 +/- 0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4 +/- 18.2 (86.1 +/- 13.2) fmol/mg protein and a Kd of 10.5 +/- 2.9 (15.1 +/- 3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4 +/- 13.9 (35.9 +/- 8.2) fmol/mg protein and a Kd of 4.1 +/- 1.2 (1.7 +/- 1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3 +/- 42.1 (142.9 +/- 17.8) fmol/mg protein and a Kd of 16.3 +/- 4.9 (9.2 +/- 7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4 +/- 51.9 (38.8 +/- 7.4) fmol/mg protein and a Kd of 16.2 +/- 3.2 (2.5 +/- 1.2) nM. It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are able to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Mineralocorticoids upregulate arterial contraction to epidermal growth factor

The present studies test the hypothesis that contraction to EGF is dependent on mineralocorticoids and/or an elevation in systolic blood pressure (SBP). Endothelium-denuded thoracic aortas from sham normotensive, N(omega)-nitro-L-arginine (L-NNA) hypertensive, Wistar-Kyoto (WKY), and spontaneously hypertensive rats (SHR) were used in isolated tissue-bath experiments. Maximal contraction to epidermal growth factor [EGF; percentage of phenylephrine (PE; 10 umol/l)-induced contraction] was greater in strips from L-NNA (32 +/- 5%) and SHR (53 +/- 8%) rats compared with sham and WKY rats (17 +/- 1 and 12 +/- 4%, respectively). Wistar-Furth rats became only mildly hypertensive when given DOCA salt (134 +/- 6 mmHg) compared with Wistar rats (176 +/- 9 mmHg), but aortas from both strains had a similarly enhanced contraction to EGF (approximately 9 times the maximal contraction of sham aorta). Furthermore, in vitro incubation of aortas from Wistar and Wistar-Furth rats with aldosterone (10 nmol/l) increased EGF-receptor mRNA expression by >50%. These data indicate that arterial contraction to EGF may occur independent of hypertension and be stimulated by mineralocorticoids.     

High affinity renal [3H]flunitrazepam binding: Characterization,localization, and alteration in hypertension

The binding of [³H]flunitrazepam was studied in membranes prepared from the kidney and cerebral cortex of unilaterally nephrectomized rats made hypertensive by simultaneous deoxycorticosterone acetate (DOCA) and NaCl administration. A significant 35–43% increase in the number of [³H]flunitrazepam binding sites (Bmax) was found in the renal membranes prepared from the hypertensive rats; there was no change in the density of binding sites in the membranes obtained from the cerebral cortex. The K_d of [³H]flunitrazepam binding did not change either in the renal or in the cerebral membranes (～ 12 nM in the kidney and ～2.0 nM in the brain). Drug specificity studies with renal membranes showed that the inhibition of [³H]flunitrazepam binding by various benzodiazepines did not jibe with their pharmacologic potency as anxiolytic agents. An intrarenal distribution of specific [³H]flunitrazepam binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. The evidence that the renal benzodiazepine binding site is of high affinity, is specific, has a unique distribution, and is regulated during hypertension suggests that it may be associated with an important pathophysiologic structure.     

Ligand binding profiles of delta-opioid receptor in human cerebral cortex membranes: evidence of delta-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.     

[3H]Dipyridamole Binding to Guinea Pig Brain Membranes: Possible Heterogeneity of Central Adenosine Uptake Sites

The binding of [3H]dipyridamole ([3H]DPR) to guinea pig brain membranes is described and compared to that of [3H]nitrobenzylthioinosine ([3H]NBI). The binding of [3H]DPR is saturable, reversible, and specific with pharmacologic evidence indicating that this ligand is binding to the adenosine uptake site. Compared to [3H]NBI the binding of [3H]DPR is of higher capacity (Bmax = 208 +/- 16 fmol/mg protein for [3H]NBI and 530 +/- 40 fmol/mg protein for [3H]DPR) and lower affinity (KD = 0.35 +/- 0.02 nM for [3H]NBI and 7.6 +/- 0.7 nM for [3H]DPR). The adenosine uptake inhibitors are the most potent inhibitors of binding (Ki of 10(-8)-10(-7) M) whereas adenosine receptor ligands such as cyclohexyladenosine, 2-chloroadenosine, and various methylxanthines are several orders of magnitude less potent (Ki 10(-5)-10(-2). The inhibition of [3H]DPR binding by NBI is biphasic, with only 40% of binding being susceptible to inhibition of NBI concentrations less than 10(-5) M. The tissue distribution of [3H]DPR binding parallels that of [3H]NBI although in most cases significantly more sites are observed with [3H]DPR. Calcium channel blocking agents such as nifedipine, nimodipine, and verapamil are also inhibitors of [3H]DPR binding with potencies in the micromolar range. The data are consistent with [3H]DPR being a useful additional ligand for the adenosine uptake site and provide evidence that multiple uptake binding sites exist of which only about 40% are NBI-sensitive.     

Characterization and Regional Distribution of α2-Adrenoceptors in Postmortem Human Brain Using the Full Agonist [3H]UK 14304

The full agonist [3H]UK 14304 [5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline] was used to characterize alpha 2-adrenoceptors in postmortem human brain. The binding at 25 degrees C was rapid (t1/2, 4.6 min) and reversible (t1/2, 14.1 min), and the KD determined from the kinetic studies was 0.48 nM. In frontal cortex, the rank order of potency of adrenergic drugs competing with [3H]UK 14304 or [3H]clonidine showed the specificity for an alpha 2A-adrenoceptor: UK 14304 approximately equal to yohimbine approximately equal to oxymetazoline approximately equal to clonidine greater than phentolamine approximately equal to (-)-adrenaline greater than idazoxan approximately equal to (-)-noradrenaline greater than phenylephrine greater than (+/-)-adrenaline much greater than corynanthine greater than prazosin much greater than (+/-)-propranolol. GTP induced a threefold decrease in the affinity of [3H]UK 14304, with no alteration in the maximum number of binding sites, suggesting that the radioligand labelled the high-affinity state of the alpha 2-adrenoceptor. In the frontal cortex, analyses of saturation curves indicated the existence of a single population of noninteracting sites for [3H]UK 14304 (KD = 0.35 +/- 0.13 nM; Bmax = 74 +/- 9 fmol/mg of protein). In other brain regions (hypothalamus, hippocampus, cerebellum, brainstem, caudate nucleus, and amygdala) the Bmax ranged from 68 +/- 7 to 28 +/- 4 fmol/mg of protein. No significant changes in the KD values were found in the different regions examined. The binding of [3H]UK 14304 was not affected by age, sex or postmortem delay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in 5-HT(1A) receptors and adenylyl cyclase response by trazodone in regions of rat brain

The in vivo effect of trazodone on the density of [(3)H]5-HT binding sites and 5-HT(1A) receptors and adenylyl cyclase (AC) response was studied in regions of rat brain. The chronic administration of trazodone (10 mg/Kg body wt, 40 days) resulted in a significant downregulation of [(3)H]5-HT binding sites and 5-HT(1A) receptors in cortex and hippocampus. Trazodone significantly (p < 0.0001) decreased the density of [(3)H]5-HT binding sites in cortex (42.6 +/- 3.6 fmol/mg protein, 65%) and hippocampus (12.6 +/- 1.6 fmol/mg protein, 87%) when compared to control values of 121.9 +/- 5.4 and 99.3 +/- 7.5 fmol/mg protein in these regions, respectively. Similarly there was a significant (p < 0.0001) decrease in the density of 5-HT(1A) receptors in both cortex (7.2 +/- 0.5 fmol/mg protein, 70%) and hippocampus (6.3 +/- 1.2 fmol/mg protein, 79%) when compared to control values of 24.2 +/- 2.1 and 30.6 +/- 3.7 fmol/mg protein, in these regions respectively. However, the affinity of [(3)H]5-HT to 5-HT binding sites (1.83 +/- 0.26 nM, p < 0.0001) and [(3)H]8-OH-DPAT to 5-HT(1A) receptors (0.60 +/- 0.06 nM, p < 0.05) was significantly decreased only in cortex when compared to the control K(d) values of 0.88 +/- 0.04 nM and 0.47 +/- 0.02 nM in these regions, respectively.The basal AC activity did not alter in treated rats, where as, the inhibition of forskolin-stimulated AC activity by 5-HT (10 microM) was significantly (p < 0.0001) decreased both in cortex (43%) and hippocampus (40%) when compared to control levels. In conclusion, chronic treatment with trazodone results in downregulation of 5-HT(1A) receptors in cortex and hippocampus along with concomitant increased AC response, suggesting the involvement of 5-HT(1A) receptor-mediated AC response in the mechanism of action of trazodone.     

Dihydropyridine [methyl-3H]PN 200–110 Binding and Myogenesis in Intact Muscle Cells In Vitro

The radioligand dihydropyridine [methyl-3H]PN 200-110 binds to contracting myotubes in culture derived from chick embryo pectoralis muscle. [methyl-3H]PN 200-110 binds specifically to high-affinity sites, with nonspecific binding only between 15 and 30% of the total binding. A Scatchard plot of the specific binding revealed a single high-affinity binding site with a KD (dissociation constant) of 0.5 nM +/- 0.2 nM and Bmax (number of binding sites) of 100 fmol/10(6) nuclei. We employed this sensitive assay to probe the appearance of high-affinity [methyl-3H]PN 200-110 binding sites during myogenesis. The time course of appearance of high-affinity binding sites lags behind that of fusion. Low-calcium media prevented the differentiation of myoblasts and blocked the appearance of high-affinity sites. Chelation of intracellular calcium before or after fusion of myoblasts with the calcium indicator Quin 2 prevented the appearance of dihydropyridine binding sites. These findings are consistent with the view that the expression of dihydropyridine receptors is modulated by the intracellular calcium.     

Opposite changes in imidazoline I2 receptors and alpha2-adrenoceptors density in rat frontal cortex after induced gliosis

Opposite age-dependent changes in alpha2-adrenoceptor and imidazoline I2 receptor (I2-IRs) density have been related to brain gliosis development with aging. To check this hypothesis we applied in rats a model of reactive gliosis induced by heat. The specific binding of [3H]idazoxan (0.5-20 nM) in the presence of (-)adrenaline (5 x 10(-6) M) to membranes from rat brain cortex showed that the density of I(2)-IRs was significantly higher in membranes of injured cortex (Bmax=60+/-6 fmol/mg protein; n=9) than in control (Bmax=38+/-3 fmol/mg protein; n=9; p=0.0053). Conversely, the density of alpha2-adrenoceptors, measured by [3H]clonidine (0.25-16 nM), in the injured cortex (Bmax=75+/-4 fmol/mg protein; n=9) was significantly lower than in sham membranes (Bmax=103+/-7 fmol/mg protein; n=9; p=0.0035). No significant differences in receptor's affinity were observed between both groups. These results support the hypothesis that gliosis induces opposite changes in alpha2-adrenoceptor and I2-IR density.