3H]dihydroalprenolol binding in the rat brainstem

The binding of [3H]dihydroalprenolol to beta-adrenergic receptors in the rat brainstem was studied. Propranolol inhibited dihydroalprenolol binding to a greater extent than did isoproterenol or epinephrine. Scatchard plots obtained with propranolol were biphasic whereas those obtained with isoproterenol and epinephrine were monophasic. The results indicate that in the brainstem propranolol displaces dihydroalprenolol from sites other than beta-adrenergic receptors. Nonspecific binding of dihydroalprenolol in this tissue therefore cannot be assayed using propranolol but should be measured with isoproterenol or epinephrine.     

Serotonin depletion unmasks serotonergic component of [3H]dihydroalprenolol binding in rat brain

Selective lesions of serotonin neurons or inhibition of serotonin synthesis results in an increase in the number of [3H]dihydroalprenolol binding sites in several areas of rat brain. Previously, this increase in binding sites was interpreted as an increase in beta-adrenergic receptors. However, the lesion-induced increase in [3H]dihydroalprenolol is not accompanied by an increase in isoproterenol- or norepinephrine-stimulated cyclic AMP production. The increased binding of [3H]dihydroalprenolol is blocked by the addition of serotonin but not by the addition of norepinephrine or dopamine to the assays. Furthermore, the addition of metergoline, a serotonin antagonist, also blocked the increase in lesioned tissues. Thus, the lesion-induced increase in [3H]dihydroalprenolol binding appears to represent an increase in serotonergic binding sites. Among drugs with some selectivity for serotonin-1 receptor subtypes, trifluoromethylphenylpiperazine and RU-24929 were as effective as serotonin in blocking lesion-induced increases in [3H]dihydroalprenolol binding. However, 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT), spiperone, and mesulergine were either much less effective than serotonin or completely ineffective. Radioligand binding to serotonin-1A and serotonin-1B sites with [3H]8-OH-DPAT and 125I-cyanopindolol, respectively, after lesions of serotonin axons or depletion of serotonin was not increased, despite a marked increase in [3H]dihydroalprenolol binding in the same tissues. When tissues from control rats or rats with serotonin lesions were preincubated at 37 degrees for 10 min to remove endogenous serotonin bound to receptors, the binding of [3H]dihydroalprenolol in controls was increased to the level seen in lesioned tissues. Thus, [3H]dihydroalprenolol binds primarily to beta-adrenergic receptors in control membranes that are not preincubated; however, either preincubation of control tissues or serotonin depletion unmasks a serotonin-1 receptor subtype to which [3H]dihydroalprenolol binds in addition to the beta-adrenergic receptor.     

Temperature dependence of [3H]PAF binding to washed human platelets.

Our results indicate that the binding of [3H]PAF to a single class of high affinity sites on human platelets is endothermic and largely entropy-driven. The increase in entropy can be attributed to the disorganization of water molecules bound to both receptor and ligand during the binding process.     

Chronic treatment with nifedipine does not change the number of [3H]nitrendipine and [3H]dihydroalprenolol binding sites

Possible receptor changes occurring after withdrawal of chronic nifedipine treatment or chronic propranolol treatment were examined by administering nifedipine (100 mg/kg per day) or propranolol (45 mg/kg per day) to rats for 2 weeks and then withdrawing treatment [3H]Nitrendipine and [3H]DHA binding were measured in membrane fragments of the ventricle. In propranolol-treated rats, 8 h after the last administration, the maximum binding for [3H]DHA was significantly increased from 79.9 +/- 8.0 to 139.8 +/- 12.8 fmol/mg protein (mean +/- S.E.); the dissociation constant was significantly increased from 4.9 +/- 0.7 to 10.7 +/- 1.2 nM. On the other hand, in nifedipine-treated rats, 12 and 48 h after the last administration, [3H]nitrendipine binding and [3H]DHA binding had not changed significantly. These results indicate that the mechanism of the calcium antagonist withdrawal syndrome may be different from that of beta-blocker withdrawal syndrome.     

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

[3H]Dihydroalprenolol ([3H]DHA) specific binding (determined by the difference in the presence and absence of 20 microM (-)isoprenaline) to rat colon membranes was saturable (Bmax = 39.6 fmol/mg protein), of high affinity (Kd = 0.87 nM) and stereospecific (IC50 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 (Ki range 1.9-3.3 nM) by the non-selective beta-adrenoceptor antagonists pindolol, alprenolol, but not by the non-adrenergic compounds 5-hydroxytryptamine, 8-hydroxydipropylaminotetraline, methysergide, dopamine and verapamil (Ki greater than 10,000 nM). The selective beta 1- and beta 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 (beta 2) and 25% (beta 1) of total sites. The relative competing potencies of reference adrenergic agonists also suggested a prevalence of beta 2-adrenergic sites. The new agonists phenylethanolaminotetralines (PEATs), highly selective for the atypical beta-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 Kd for high affinity sites (beta 1 and beta 2), was prevented by saturating concentrations of isoprenaline. Under this condition, the representative PEAT, SR 58611A, highly potent and selective for atypical beta-adrenoceptors in functional tests, and its pharmacologically inactive enantiomer, both inhibited the residual binding equipotently. In conclusion, [3H]DHA binding did not detect atypical beta-adrenoceptor sites in rat colon membranes, most probably because of its weaker affinity for them than for the coexisting beta 1 and beta 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.     

Age-related changes in [3H]prazosin binding and phosphoinositide hydrolysis in rat ventricular myocardium

1. We investigated, using rats at 2, 12 and 24 months of age, the effect of aging on ventricular alpha 1-adrenoceptor responsiveness by measuring the [3H]prazosin binding, [3H]inositol phosphate formation, the cholesterol/phospholipid ratio and membrane viscosity. 2. The density of specific [3H]prazosin binding sites on membranes was significantly reduced with advanced age, and the binding affinity for [3H]prazosin was significantly increased. 3. Norepinephrine (NE)-induced formation of [3H]inositol phosphates in slices was also reduced with advanced age. However, the EC50 for NE stimulation of [3H]inositol phosphates was significantly higher when compared to those at adult ages. 4. The cholesterol content in membranes was significantly decreased with advanced age; concomitantly, a decrease in the cholesterol/phospholipid ratio was observed. The membrane viscosity as measured by using 1,6-diphenyl-1,3,5-hexatriene significantly decreased with advanced age. 5. These results suggest that the altered cholesterol/phospholipid ratio and/or the membrane viscosity during aging may account for the increase in the binding affinity for [3H]prazosin and/or the reduced responsiveness of aged ventricular myocardium to NE.     

3H]glycogenolysis in brain slices mediated by beta-adrenoceptors: comparison of physiological response and [3H]dihydroalprenolol binding parameters

The subclass of beta-adrenergic receptors mediating glycogenolysis in slices from cerebral cortex of the mouse, incubated in the presence of [3H]glucose, was identified by comparing the relative potencies of agonists and the inhibition constants of antagonists to those found on reference systems. (+/-)Isoprenaline, (-)adrenaline and (-)noradrenaline produced a concentration-related glycogenolysis with Kact values of 2.2 x 10(-8) M, 2.8 x 10(-7) M and 3.6 x 10(-7) M, respectively. Zinterol, a selective beta 2-adrenergic agonist, did not produce any glycogenolytic response even in a large concentration. Salbutamol, a predominantly beta 2-adrenergic receptor agonist, elicited in a very large concentration (10(-4) M) less than 40% glycogenolysis, an effect which was not related to stimulation of beta-adrenergic receptors. The predominantly beta 1-adrenergic receptor antagonists, practolol and metoprolol shifted the concentration-response curve to noradrenaline to the right, with apparent Ki values of 8.0 x 10(-7) M and 7.6 x 10(-8) M, respectively, close to those reported in the rat heart. These various data indicate that the glycogenolytic response is selectively mediated by beta 1-adrenergic receptors. Under experimental conditions which were strictly identical to those used to measure glycogenolysis, a saturable binding of [3H]dihydroalprenolol to the slices occurred with Kd and Bmax values consistent with corresponding values on cell free preparations. Whereas the Ki values of antagonists were similar on the two systems, the Kact values of agonists on glycogenolysis were 10 times less than the Ki values for the binding of [3H]dihydroalprenolol. This suggests that the maximal glycogenolytic response is elicited for a partial receptor occupancy.     

Beta-adrenergic receptors in guinea-pig liver plasma membranes and thermal lability of [3H]dihydroalprenolol binding sites

beta-Adrenergic receptors in guinea-pig liver plasma membranes were characterized by radioligand binding, using l-[3H]dihydroalprenolol ([3H]DHA), l-3-[125I]iodocyanopindolol ([125I]CYP) and dl-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2- one hydrochloride [( 3H]CGP-12177). The binding of both [125I]CYP and [3H]CGP-12177 to membranes exhibited high affinity (Kd = 3.5 +/- 0.2 pM for [125I]CYP and 0.75 +/- 0.10 nM for [3H]CGP-12177) and stereospecificity; the maximal binding sites were 130 +/- 15 and 137 +/- 8 fmoles/mg protein respectively. Catecholaminergic agonists competed for these binding sites in the order l-isoproterenol greater than l-epinephrine greater than l-norepinephrine, which is typical for beta 2-adrenergic receptors. The binding data are supported by parallel experiments on adenylate cyclase activation by catecholamines, and on antagonism of this activation by beta 1- and beta 2-selective blockers. The binding of [3H]DHA was excessive (Bmax = 21.4 pmoles/mg protein), exhibited low affinity (Kd = 34.6 nM), and lacked stereospecificity. When liver membranes were incubated at 50 degrees for 40 min in the presence of an agonist, l-isoproterenol, the binding of [3H]DHA to the heat-treated membranes exhibited high affinity (Kd = 1.07 +/- 0.17 nM) and the Bmax was reduced to 139 +/- 22 fmoles/mg protein. In such membranes, as opposed to native membranes, stereospecificity was evident and catecholaminergic agonists competed for the binding sites in the order typical for beta 2-adrenergic receptors. However, agonist competition of the binding to the heat-treated membranes could not be modulated by guanine nucleotides, indicating a loss of communication between the receptor and the guanine nucleotide regulatory protein.     

Melatonin enhancement of [3H]-gamma-aminobutyric acid and [3H]muscimol binding in rat brain

The pineal hormone, melatonin, enhanced the sodium-independent binding of [3H]-gamma-aminobutyric acid ([3H]GABA) and [3H]muscimol in the rat cerebral cortex in vitro. This effect was augmented by preincubation of synaptic membranes with melatonin but was abolished by preincubation with Triton X-100. Saturation binding studies using [3H]GABA (2.5 to 1000 nM) indicated that the melatonin-induced enhancement of binding is due to an increase in low-affinity GABAA binding sites. These findings suggest that the central effects of melatonin involve modulation of GABAergic function.     

Temperature dependence of the binding of [3H]mepyramine and related compounds to the histamine H1 receptor

The extent of the promethazine-sensitive binding of [3H]mepyramine to a washed fraction from guinea pig cerebellum was little altered between 4 degrees and 30 degrees. The dissociation of [3H]mepyramine from the H1 receptor was markedly temperature-sensitive. An Arrhenius plot of the variation of the dissociation rate constant, k-1, with temperature was linear over the range 37 degrees -15 degrees (E alpha = 160 kJ mole-1). The association rate constant, k1, was also temperature-dependent, and an Arrhenius plot approximated well to a straight line between 30 degrees and 15 degrees (E alpha = 112 kJ mole-1). The linear relationship may hold down to 4 degrees. A consequence of the slow dissociation at 4 degrees is that the IC50 for mepyramine inhibition of promethazine-sensitive [3H]mepyramine binding at 4 degrees is independent of the concentration of [3H]mepyramine if the cerebellar homogenate is first incubated with the nonradioactive antagonist before addition of the 3H-ligand. Promethazine binding showed a temperature dependence similar to that of mepyramine, and for both antagonists and for chlorpromazine the affinity constant was not greatly increased (equal to or less than a factor of 2) at 4 degrees compared with 30 degrees. Tripelennamine showed a more rapid dissociation at 4 degrees than the other antagonists. Mequitazine dissociated slowly at 4 degrees, but the affinity constant was lower at 4 degrees than at 30 degrees.     

Acetylcholinesterase potentiates [3H]fluorowillardiine and [3H]AMPA binding to rat cortical membranes

In addition to its action at cholinergic synapses acetylcholinesterase (AChE) has been proposed to modulate neuronal activity by mechanisms unrelated to the hydrolysis of acetylcholine. We have investigated the effects of AChE on the binding of the specific AMPA receptor agonists (S)-[3H]5-fluorowillardiine ([3H]FW) and [3H]AMPA to rat cortical membranes. Pretreatment of membranes with AChE causes a dose-dependent increase in the binding of both radiolabelled agonists with a maximal increase to approximately 60% above control. This increase is completely blocked by the specific AChE inhibitors propidium, physostigmine, DFP and BW 284C51. AChE pretreatment had no effect on [3H]kainate binding. [3H]FW binding to membranes from young (15-day-old) rats is four orders of magnitude more sensitive to AChE modulation than membranes from adult rats (EC50 values of 4x10(-5) and 0.1 unit/ml, respectively) although the total percentage increase in binding is similar. Furthermore, the AChE-induced potentiation of [3H]FW binding is Ca2+ - and temperature-dependent suggesting an enzymatic action for AChE in this system. Saturation binding experiments with [3H]FW to adult membranes reveal high and low affinity binding sites and demonstrate that the main action of AChE is to increase the Bmax of both sites. These findings suggest that modulation of AMPA receptors could provide a molecular mechanism of action for the previously reported effects of AChE in synapse formation, synaptic plasticity and neurodegeneration.     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

Characteristics of [3H]sultopride binding to rat brain

The binding of [3H]sultopride, a benzamide drug, to rat brain was investigated in vitro. Specific [3H]sultopride binding was observed in dopaminergic regions: striatum, nucleus accumbens, olfactory tubercle, substantia nigra, frontal cortex and anterior pituitary. Specific [3H]sultopride binding to striatum was saturable and had one high affinity binding site with a KD of 5.8 nM and a total density of receptors 25.7 pmol/g. [3H]Sultopride binding was stereoselectively displaced by (-)- and (+)-sultopride. Inhibition studies indicated that all neuroleptic drugs and dopamine were capable of displacing sultopride from its binding sites. A highly significant correlation was observed between IC50 values against [3H]sultopride and those against [3H]spiperone binding. Specific [3H]sultopride binding was highly dependent on the presence of sodium ions. The results suggest that the characteristics of sultopride binding sites seem to be similar to those of the D2-receptor labeled by spiperone and haloperidol. The sultopride binding site was highly dependent on the presence of sodium ions and may thus be characterized as a sodium-dependent D2-receptor.