CHARACTERIZATION OF BINDING SITES FOR [3H]-IDAZOXAN, [3H]-P-AMINOCLONIDINE AND [3H]-RAUWOLSCINE IN THE KIDNEY OF THE DOG

1. We characterized the binding of [³H]-rauwolscine, [³H]-p-aminoclonidine and [³H]-idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the α₂-adrenoceptor- and imidazoline-preferring binding sites in this organ. 2. [³H]-Rauwolscine bound to an apparent single site with an affinity (K_D) of 2.2 nmol/ L and a maximum density (B_max) of 58.5 fmol/mg protein, when 10 μmol/L idazoxan defined non-specific binding. However displacement studies demonstrated that a number of compounds, including prazosin, inhibited [³H]-rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [³H]-rauwolscine binding sites had a relatively low affinity for prazosin (K_I= 398 nmol/L), while the remainder had a relatively high affinity for prazosin (K_I= 7.9 nmol/ L). 3. [³H]-p-Aminoclonidine bound to an apparent single site (K_D= 5.2 nmol/L; B_max= 72.4 fmol/mg protein), when 10 μmol/L phentolamine defined non-specific binding. When 1 μmol/L of the potent and selective α₂-adrenoceptor antagonist 2-methoxyidazoxan was included in the incubate, no specific binding was detected. We therefore conclude that under the conditions of this experiment [³H]-p-aminoclonidine binds only to α₂-adrenoceptors in the dog kidney. 4. [³H]-Idazoxan bound to two sites, with a higher (K_D= 0.95 nmol/L; B_max= 43.9 fmol/mg protein) and lower (K_D= 9.1 nmol/L; B_max= 93.8 fmol/mg protein) affinity, respectively, when 1 mmol/L phentolamine defined non-specific binding. When 10 μmol/ L GTPγS was included in the incubate, the low affinity site was unaffected but the maximum binding at the higher affinity site was reduced by 79%. 2-Methoxyidazoxan displaced [³H]-idazoxan in a monophasic manner and with low potency (IG₅₀=11.5 μmol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displaced [³H]-idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney contains a heterogeneous population of α₂-adrenoceptors that can be labelled either with [³H]-rauwolscine or [³H]-p-aminoclonidine. The dog kidney also contains a heterogeneous population of non-adrenoceptor imidazoline-preferring binding sites of the I₂-subtype, that can be labelled with [³H]-idazoxan. The binding site for which [³H]-idazoxan has the highest affinity appears to be coupled to a guanine nucleotide binding regulatory protein.     

Two distinct α1-adrenergic receptor sites in rat liver: Differential binding of (−)-[3H]norepinephrine, [3H]prazosin and [3H]dihydroergocryptine: Effects of guanine nucleotides and proteolysis; implications for a two-site model of α-receptor regulation

(?)-[³H]Norepinephrine, [³H]prazosin and [³H]dihydroergocryptine bind to rat liver plasma membranes in a manner indicating a selective interaction with α₁-adrenergic receptors. All three ligands display monophasic saturation with a single component on Scatchard analysis. The binding capacities of (?)-[³H]norepinephrine, [³H]prazosin and [³H]dihydroergocryptine are 340 ± 70 fmol/mg of protein 760 ± 40 fmol/mg of protein and 1200 ± 300 fmol/mg of protein, respectively. Differential drug potencies in competing for (?)-[³H]norepinephrine and [³H]prazosin binding sites suggest that these two ligands label two distinct binding sites at the α₁-adrenergic receptor, while [³H]dihydroergocryptine labels both sites. Guanine nucleotides lower the apparent affinity of (?)-[³H]norepinephrine for its binding site, without affecting the number of sites or the binding of [³H]prazosin and [³H]dihydroergocryptine. Incubation of membranes with α-chymotrypsin slightly reduces the binding of [³H]prazosin and [³H]dihydroergocryptine, but causes a 2-fold increase in (?)-[³H]norepinephrine binding. Both the number of (?)-[³H]norepinephrine binding sites and the affinity are increased. Following proteolysis, (?)-[³H]norepinephrine binding still occurs to a single class of sites, but is no longer affected by guanine nucleotides. The effect of α-chymotrypsin is abolished by pretreatment of membranes with the irreversible α-adrenergic antagonist phenoxybenzamine. We propose that in rat liver, (?)-[³H]norepinephrine labels the physiologically active form of the α-adrenergic receptor while [³H]prazosin binds to a precursor form of the active receptor, and/or to an α₁-adrenergic binding site not coupled to an effector system.     

POSITIVE CHRONOTROPIC REPONSES TO CARDIAC α1-ADRENORECEPTOR ACTIVATION IN THE PITHED RAT

• 1 Adrenaline (0.1–10 μg/kg), noradrenaline (0.1–10 μg/kg) and phenylephrine (1–100 μg/kg) acted on both cardiac α₁- and β-adrenoreceptors to induce positive chronotropic responses in the pithed rat.
• 2 When β-adrenoreceptors were blocked by propranolol (1 mg/kg), the residual chronotropic responses were due to activation of α₁-adrenoreceptors since they were significantly reduced by prazosin (10–100 μg/kg).
• 3 Methoxamine (10–300 μg/kg) acted solely on cardiac α₁-adrenoreceptors to induce positive chronotropic responses which were abolished by prazosin (10–100 μg/kg) alone, as has been demonstrated previously for amidephrine.
• 4 The rank order of potency for eliciting the positive chronotropic response to α₁-adrenoreceptor activation was adrenaline > noradrenaline > phenylephrine > methoxamine.
• 5 The positive chronotropic responses to adrenaline (3–10 μg/kg), noradrenaline (3–10 μg/kg) and phenylephrine (30–100 μg/kg) produced by activating α₁-adrenoceptors had a slower time course than did the chronotropic responses produced by activation of β-adrenoreceptors.

     

K+ release from rat parotid cells: An α1-adrenergic mediated event

To characterize α-adrenergic receptors in rat parotid gland tissue, 9,10-[9,10-³H(N)]-dihydro-α-ergocryptine ([³h]dhe) and [³H]prazosin binding to membranes and stimulated K⁺ release from parotid cell aggregates were examined. Prazosin (selective α₁-adrenergic antagonist), displacement of [³H]DHE binding from parotid membranes was biphasic, indicating the presence of both α₁- and α₂-adrenergic receptors. The numbers of α₁- and α₂-receptors were about equal. α₁-Adrenergic receptors were further studied by [³H]prazosin binding. [³H]Prazosin binding was a rapid, reversible, saturable and stereospecific process, with high affinity (K_D = 0.38 nM) and low capacity (B_max = 380 fmoles ligand bound/g tissue, 10.1 fmoles/mg protein) as determined by Scatchard analysis. The characteristics of [³H]prazosin binding were in good agreement with those of catecholamine-stimulated K⁺ release, suggesting that K⁺ release from rat parotid gland cells is an α₁-adrenergic mediated effect.     

EFFECT OF THYROID STATUS ON β-ADRENORECEPTORS AND MUSCARINIC RECEPTORS IN THE RAT LUNG

• 1 The effects of thyroid status on the specific binding of the muscarinic ligand (–)-[³H] quinuclidinyl benzilate (QNB) and of the β-adrenoreceptor ligand (–)-[³H] dihydroalprenolol (DHA) in the adult rat lung were investigated.
• 2 The specific binding of (–)-[³H] quinuclidinyl benzilate (QNB) to lung membranes was saturable and the equilibrium dissociation constant (K_D) determined from Scatchard analysis was 54 pM. Kinetic analysis of the binding of [³H] QNB yielded a K_D of 42 pM. [³H] QNB binding was inhibited by muscarinic agonists and antagonists, the order of their potency was l-hyoscyamine>atropine>scopolamine>oxotremorine>carbachol. These data were consistent with [³H] QNB binding to the muscarinic receptor.
• 3 Adult male rats treated for 2 weeks with the antithyroid agent 3-amino-1,2,4-triazole (ATZ) showed a 52% and 80% reduction in the serum concentration of triiodothyronine (T₃) and thyroxine (T₄) respectively. These hypothyroid rats also had a 39% decrease in the concentration of lung β-adrenoreceptors and a 37% decrease in the concentration of lung muscarinic receptors as compared to euthyroid controls. Concurrent treatment of rats with ATZ and T₄ for 2 weeks resulted in a reduction of 15% and 20% in the concentration of lung β-adrenoreceptors and muscarinic receptors respectively. The K_D values for [³H] DHA and [³H] QNB binding did not change with the ATZ or ATZ + T₄ treated groups.
• 4 Administration of T₄ (500 μg/kg/day) to male rats for 12 days did not result in any significant change in the concentration of either β-adrenoreceptors or muscarinic receptors compared to euthyroid controls. No change in the K_K values for [³H] DHA or [³H] QNB binding were detected.
• 5 The results show that hypothyroid rats have a reduced lung concentration of both β-adrenoreceptors and muscarinic receptors whereas in hyperthyroid rats these receptors do not significantly change from euthyroid controls.

     

Comparison of the Binding Activities of Some Drugs on α2A, α2B and α2C-Adrenoceptors and Non-Adrenergic Imidazoline Sites in the Guinea Pig

Abstract: Simultaneous computer modelling of control and guanfacine-masked [³H]-MK 912 saturation curves as well as guanfacine competition curves revealed that both α_2A- and α_2C-adrenoceptor subtypes were present in the guinea pig cerebral cortex. The K_d value of [₃H]-MK 912 determined for the α_2A-subtype was 403 pM and for the α_2C-subtype 79.8 pM; the receptor sites showing capacities 172 and 19.5 fmol/mg protein, respectively. The K_ds of guanfacine were 20 and 880 nM for the α_2A- and α_2C-adrenoceptor, respectively. In the guinea pig kidney [₃H]-MK 912 bound to a single saturable site with K_d 8.34 nM and capacity 285 fmol/mg protein, the site showing pharmacological properties like an α_2B-adrenoceptor. Binding constants of 22 compounds for the three guinea pig α₂-adrenoceptor subtypes were determined by computer modelling competition curves using for the cerebral cortex a “3-curve assay”, for the kidney an “1-curve assay”, and using [³H]-MK 912 as labelled ligand. Of the tested drugs guanfacine and BRL 44408 were found to be clearly α_2A-selective. Spiroxatrine, yohimbine, rauwolscine and WB 4101, as well as [₃H]-MK 912 itself, were found to be α_2C-selective. The most selective compounds for α_2B-adrenoceptors, when compared to α_2A-adrenoceptors, were ARC 239 and prazosin. In the guinea pig kidney [₃H]-p-aminoclonidine bound to α₂-adrenoceptors as well as to non-adrenergic imidazoline sites. The α₂-adrenoceptors could be completely blocked using 10 μM (-)-adrenaline without the non-adrenergic sites being affected. During these conditions the analysis of combined saturation and competition studies using labelled and unlabeled p-aminoclonidine with computer modelling revealed that the ligand labelled two different sites with K_ds of 310 and 47,000 nM, respectively. Competition curves of 16 compounds for the non-adrenergic [₃H]-p-aminoclonidine sites were shallow and resolved into two-site fits. For the high affinity [₃H]-p-aminoclonidine site the highest affinities were shown by 1-medetomidine, UK-14,304, guanabenz and detomidine; the K_ds of these drugs ranging 26–72 nM. All drugs tested showed low but varying affinities for the low affinity [₃H]-p-aminoclonidine site. These data indicated that the [³H]-p-aminoclonidine binding sites of the guinea pig kidney are grossly different from the [₃H]-idazoxan binding I₂-receptors previously demonstrated also to be present in the guinea pig kidney.     

Interaction of dihydroxy-2-aminotetralin derivatives at sites labelled with [3H]clonidine, [3H]prazosin and [3H]spiperone in rat brain membranes

The interactions of 5,6- and 6,7-dihydroxy derivatives of 2-aminotetralin with [³H]clonidine and [³H]clonidine and [³H]prazosin as well as with [³H]spiperone binding sites in rat cerebral cortex membrane preparations were investigated. The hydroxy derivatives of 2-aminotetralin tested showed significant interaction with [³H]clonidine as well as with [³H]spiperone binding sites while for [³H]prazosin binding site these agents appeared virtually inactive. For interaction with [³H]clonidine binding site 6,7-dihydroxy substitutions impart greater potency that 5,6-dihydroxy substitutions and N-alkyl substitutions either make no difference or reduce the affinity of these compounds. N-alkyl substitutions, however, markedly enhance the affinity of 5,6-dihydroxy derivatives for interactions with [³H]spiperone binding site. The results suggest that some hydroxy derivatives of aminotetralin have significant interaction with both central α₂-adrenoceptor and D₂-dopamine receptor systems.     

THE EFFECTS OF KF-4317, A NOVEL COMBINED α- and β1-ADRENORECEPTOR ANTAGONIST,ON THE RAT ISOLATED RIGHT VENTRICLE AND AORTA

• 1 The effects of KF-4317 on the accumulation of radioactivity from [³H]-noradrenaline, and on the subsequent spontaneous and noradrenergic nerve-evoked outflow of radioactivity have been investigated in the rat isolated right ventricle. In addition the effects of KF-4317 on the contractions of the electrically-driven directly muscle stimulated rat right ventricle to isoprenaline and of the rat isolated aorta to phenylephrine and 5-hydroxytryptamine are reported.
• 2 KF-4317 at 1μM had no effect on the ability of the rat right ventricle to accumulate radioactivity from [³H]-noradrenaline.
• 3 The spontaneous outflow of radioactivity, following loading of the ventricle with [³H]-noradrenaline, was increased by KF-4317 at 1μM by a cocaine-insensitive mechanism.
• 4 KF-4317 at 1μM had no effect on the noradrenergic nerve-evoked outflow or radioactivity, and therefore is not an α₂-adrenoreceptor antagonist, but reduced the associated contractile response probably mainly by acting as an antagonist at postjunctional β₁-adrenoreceptors.
• 5 KF-4317 caused a parallel rightward shift of the concentration-response curve of the electrically-driven directly muscle stimulated rat right ventricle to isoprenaline. However the inhibitory effect, × 9.0 and × 237.2 in the presence of 0.1 and 1μM KF-4317, was not closely concentration-related. At 1μM, KF-4317 also depressed the maximum responses to isoprenaline. This suggests that in addition to β₁-adrenoreceptor antagonism, KF-4317 probably exerts membrane stabilizing activity.
• 6 The responses of the rat isolated aorta to phenylephrine were inhibited in a non-concentration related manner by KF-4317. Thus the responses were inhibited × 1.8, × 2.7 and × 4.7 in the presence of KF-4317 at 0.1, 1 and 1μM, respectively. The responses of the aorta to 5-hydroxytryptamine were inhibited to a small extent by KF-4317 at 1μM but potentiated to a small degree by KF-4317 at 10μM. We suggest that KF-4317 is an antagonist at α₁-adrenoreceptors and 5-hydroxytryptamine receptors but that it also has an additional postjunctional action, in the rat aorta, to increase the sensitivity to both phenylephrine and 5-hydroxytryptamine.

     

EVALUATION OF THE SELECTIVITY OF α-ADRENORECEPTOR BLOCKING DRUGS FOR POSTSYNAPTIC α1- AND α2-ADRENORECEPTORS IN A SIMPLE ANIMAL MODEL

• 1 The increase in diastolic pressure of pithed, normotensive rats was determined after i.v. administration of the α-adrenoreceptor agonists L-phenylephrine and B-HT 933.
• 2 α-Adrenoreceptor antagonists varied widely in their relative inhibitory effects towards either L-phenylephrine- or B-HT 933-induced vasoconstrictor responses. Prazosin displayed the highest affinity for the postsynaptic α-adrenoreceptor triggered by L-phenylephrine. The rank order of potency was further: phentolamine > dihydroergotamine > clozapine > corynanthine > azapetine > yohimbine > piperoxan > tolazoline > mianserin > rauwolscine. On the other hand, the rank order of potency towards B-HT 933 was: dihydroergotamine > rauwolscine > yohimbine > phentolamine > piperoxan > prazosin > tolazoline > mianserin > corynanthine > azapetine > clozapine. These data are in general agreement with the classification for α1-(triggered by L-phenylephrine) and α2-(triggered by B-HT 933) adrenoreceptors. Both populations are present postsynaptically in vascular smooth muscle of the pithed rat and are involved in vasoconstriction.
• 3 The ratio of K_B post α2/K_B post α1 was calculated as a measure of selectivity for either α-adrenoreceptor site. The α-adrenoreceptor antagonists used cover a 20,000-fold range of activity ratios. The antagonists most selective for either type were prazosin (α1) and rauwolscine (α2). The selectivity of the α-adrenoreceptor antagonists for postsynaptic α1- and α2-adrenoreceptors in the intact circulatory system of the pithed rat is comparable with the reported selectivity of these blocking agents for α1 (postsynaptic)- and α2 (presynaptic)-adrenoreceptors in the rabbit isolated pulmonary artery.
• 4 It is concluded that two distinct types of postsynaptic α-adrenoreceptors participate in vasoconstriction in the pithed rat. Apart from the classical α1-adrenoreceptor, vascular smooth muscle of the pithed rat contains postsynaptic α2-adrenoreceptors resembling those previously found mainly presynaptically. The presence of separate classes of postsynaptic α1- and α2-adrenoreceptors in the intact circulatory system of the pithed rat offers the possibility to use this relatively simple animal model as an in vivo test system for the pharmacological characterization of α-adrenoreceptor agonists and antagonists.

     

In vitro and in vivo approaches to the characterization of the α2-adrenoceptor

1 In order to more fully understand the role of the α₂-adrenoceptor in brain function, a combination of in vitro and in vivo techniques were utilized including radioligand binding, autoradiography, brain microdialysis and antisense oligonucleotides. 2 Binding studies showed the tritiated form of the selective α₂-adrenoceptor antagonist, RX821002 (methoxy-idazoxan) labelled an apparent single population of sites in rat brain membranes with high affinity (1 nM), for which prazosin had low affinity (1107 nM). Similar studies in rabbit brain membranes found that prazosin and oxymetazoline were able to displace. [³H]-RX821002 in a biphasic manner indicating the presence of subtypes of α₂-adrenoceptors. 3 Receptor autoradiography revealed a distribution of [³H]-RX821002 binding in rat brain consistent with the labelling of all %aL₂-adrenoceptor subtypes, namely α_2A/D-, α_2B and α_2C. 4 In rat, in vivo brain dialysis experiments demonstrated peripherally administered RX821002 elevated basal noradrenaline in frontal cortex and also, although to a lesser extent, in ventral hippocampus. RX821002 was also able to elevate extracellular dopamine in the striatum. 5 A 7-day i.c.v. infusion of an antisense oligonucleotide targeting the α_2A/D-adrenoceptor, resulted in a significant reduction in the autoradiographic density of [³H]-RX821002 binding in specific brain areas, notably the lateral septal nuclei and anterior hypothalamic area. 6 Several years of research by our group has extended our knowledge of the pharmacology and function of the α₂-adrenoceptor and has provided evidence of the roles of this receptor in the control of monoamine turnover. The successful use of antisense technology to knockdown expression of the α_2A/D subtype provides future opportunities to explore the physiology of this receptor subtype.     

Differential binding of guanabenz and its metabolites to cerebral α2-receptors: The basis for a radioligand assay specific for the drug

Guanabenz (E-2,6-dichlorobenzylideneaminoguanidine acetate, Wy-8678) and its identified metabolites were tested for their ability to displace [³H]clonidine from the cerebral α₂-receptors of rat and dog. The K_j's for inhibition of [³H]clonidine binding to rat membranes were 1.97, 0.96, 14.0, and 108 nM for clonidine, guanabenz, p-hydroxyguanabenz, and the Z-isomer of guanabenz, respectively. The other metabolites at concentrations of 10,000 nM did not displace [³H]clonidine. Scatchard analysis indicated single populations of binding sites with K_D' s of 2.37 and 2.39 nM and B_max's of 5.39 and 5.12 pmol/g for rat and dog preparations, respectively. Hill analysis yielded coefficients approximating unity, indicating a lack of cooperativity in binding. The high affinity of guanabenz relative to that of its metabolites for the α₂-receptor provided the basis for the development of an assay capable of discriminating guanabenz from its weakly hypotensive (p-hydroxyguanabenz and the Z-isomer of guanabenz) and pharmacologically inactive (2–6, dichlorobenzyl alcohol,2,6-dichlorobenzaldehyde, 2,6-dichlorobenzaldehyde azine, 2,6-dichlorobenzaldehyde semicarbizone, creatinine adduct of 2,6-dichlorobenzaldehyde, and an isomer of the creatinine adduct) metabolites. Antihypertensive agents with different mechanisms of action such as indoramin, prazosin, debrisoquine, and bethanidine bound little, if at all, and thus will not interfere with the assay of guanabenz.     

POSITIVE CHRONOTROPIC RESPONSES PRODUCED BY α-ADRENORECEPTORS IN THE PITHED RAT

• 1 Phenylephrine (1–100 γg/kg, intravenously) produced dose-dependent increases in heart rate and blood pressure in the pithed rat.
• 2 The positive chronotropic response to phenylephrine (10 μg/kg) was reduced in a dose-dependent manner by propranolol (0.01–0.3 mg/kg), but higher doses of propranolol (up to 3 mg/kg) did not reduce the response by more than about 50%. The residual response was virtually abolished by phentolamine (0.3 mg/kg) or prazosin (3 μg/kg). Labetalol (3 mg/kg) which has both α- and β-blocking activity, also abolished the positive chronotropic response.
• 3 The pressor response to phenylephrine (1–30 μg/kg) was enhanced by propranolol (1 mg/kg) and abolished by phentolamine (1 mg/kg) and prazosin (30 μg/kg). Labetalol (3 mg/kg) reduced the response to phenylephrine by 73%.
• 4 Propranolol (0.3 mg/kg) completely blocked the chronotropic and vasodepressor effects of isoprenaline (0.1 μg/kg).
• 5 It is concluded that phenylephrine acts on both α₁- and β₁-adrenoreceptors to produce an increase in heart rate, on α₁-adrenoreceptors to produce vasoconstriction and on β₂-adrenoreceptors to produce vasodilation. This latter effect is usually masked by the predominant vasoconstrictor action.

     

ANALYSIS OF THE ANTAGONISM BY PRAZOSIN OF NORADRENALINE AND PHENYLEPHRINE INDUCED CONTRACTIONS OF THE RAT ANOCOCCYGEUS MUSCLE*

• 1 Blockade of α-adrenoreceptors was analyzed with prazosin in the rat anococcygeus muscle contracted with noradrenaline (NA) and phenylephrine (PHE).
• 2 Prazosin (1.1 times 10^–8– 8.8 times 10^–7M) competitively blocked phenylephrine-induced contractions (pA₂= 8.93 ± 0.04, slope = 0.85 ± 0.07) whilst its antagonism of NA was non-competitive (pA₂= 8.45 ± 0.04, slope = 0.46 ± 0.03).
• 3 At higher concentrations, (1.76 times 10^–5– 5.28 times 10^–6M) prazosin competitively antagonized NA-induced contractions (pA₂= 7.58 ± 0.11, slope = 0.93 ± 0.09).
• 4 Phentolamine also reduced the effect of NA competitively (pA₂= 8.11 ± 0.09, slope = 0.82 ± 0.26).
• 5 It is concluded that low concentrations of prazosin selectively blocked an α₁-adrenoreceptor component of NA-mediated response whilst higher concentrations of prazosin non-selectively blocked both α₁- and α₂-adrenoreceptors.

     

Pharmacology: α-Adrenoceptor Interaction of Tetrandrine and Isotetrandrine in the Rat: Functional and Binding Assays

The action of 1S,1′S-tetrandrine, a bisbenzyltetrahydroisoquinoline alkaloid, on α₁-adrenoceptors has been compared with that of its isomer 1R,1′S-isotetrandrine. The work includes binding assays to analyse the affinity of these products for the [³H]prazosin binding site of rat cerebral cortical membranes and functional studies on rat isolated aorta to examine the effects of both alkaloids on intracellular calcium processes related or not to α-adrenoceptor activation. A radioligand receptor-binding study showed that both compounds interacted with the α₁-adrenoceptors displacing [³H]prazosin from the specific binding site. The K_i values (inhibition constants) were 0.69±0.12 and 1.6±0.4 μM for tetrandrine and isotetrandrine, respectively. The functional studies showed that both alkaloids concentration-dependently inhibited noradrenaline-induced contraction in Ca²⁺-free solution (IC50 values, i.e. the concentrations needed to induce 50% inhibition, were 252.8 and 174.9 μM for tetrandrine and isotetrandrine, respectively), the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (increase in resting tone; IC50 values 11.6 and 19.6 μM for tetrandrine and isotetrandrine, respectively) and the refilling of intracellular Ca²⁺ stores sensitive to noradrenaline (IC50 values 7.4 and 14.9 μM for tetrandrine and isotetrandrine, respectively). The results show that tetrandrine and isotetrandrine interact with α₁-adrenoceptors by displacing the [³H]prazosin binding site and that both compounds inhibit mainly the Ca²⁺-dependent process and have less action on α₁-adrenoceptors. Tetrandrine is more potent than isotetrandrine.     

Binding studies with the 5-HT1B receptor agonist [3H]CP-96,501 in brain tissues

Binding studies in rat brain membranes have shown that [³H]CP-96,501 is a selective, high affinity radioligand for 5-HT_1B receptors. Additional studies with [³H]CP-96,501 in brain tissues of several other species were undertaken to investigate its utility in detecting this receptor subtype. The presence of 5-HT_1B receptors in brain membranes of the hamster and gerbil was demonstrated with [³H]CP-96,501. This finding was confirmed by [³H]5-HT binding experiments that also indicated the presence of other 5-HT₁ subtypes (possibly 5-HT_1D, 5-HT_1E) in these species as well as the rat. Negligible specific binding of [³H]CP-96,501 was found in membranes of guinea pig brain, dog hypothalamus, bovine caudate, pig choroid plexus, and several human brain tissues, consistent with the reported absence of the 5-HT_1B receptor subtype in these species. Autoradiographic examination of rat brain sections labeled by [³H]CP-96,501 showed a dense localization in areas known to be enriched in 5-HT_1B receptors (e.g., globus pallidus, substantia nigra, superior colliculus, subiculum). On the other hand, brain sections of dog and guinea pig appeared to show no specific binding of [³H]CP-96,501 by autoradiography, in agreement with the lack of brain 5-HT_1B receptors in these tissues. © 1992 Wiley-Liss, Inc.     

Central and peripheral prazosin binding: An in vitro autoradioraphic study in the rat

Using an in vitro autoradiographic technique the binding of [³H]prazosin to brain, spinal cord, heart and vascular tissue was studied. Histologically prepared tissue sections were incubated in 5 nM [³H]prazosin or 5nM [³H]prazosin in the presence of 100 μM unlabelled prazosin base in order to establish sites exhibiting specific prazosin binding. Dense prazosin binding sites were found in all arterial tissue examined except mesenteric artery. Dense prazosin binding was also found in cardiac tissue. Prazosin binding to veins was present sometimes but often absent. Prazosin does not readily pass the blood brain barrier and in this in vitro study the only area, within the central nervous system, showing specific prazosin binding sites was the olfactory bulb. The selective binding of prazosin, an α₁-adrenoceptor antagonist, to arterial and cardiac tissue would suggest that the antihypertension induced by prazosin is mediated by a peripheral action.     

Chronic Treatment with Prazosin Causes a Subtype-specific Increase in the α1-Adrenoceptor Density of the Stressed Rat Cerebral Cortex

The effects of chronic treatment with prazosin and of immobilization stress on the α₁-adrenoceptor subtypes in rat cerebral cortex have been examined. Prazosin-treated rats were allowed free access to tap water containing two different concentrations of prazosin (16 or 156 mg L^?) for 5 weeks. The mean plasma concentrations of prazosin were 5 ng mL^? in groups treated with a low dose and 8 or 14 ng mL^? in those treated with a high dose. Immobilization stress (2 h day^?, 2 weeks) or chronic treatment with a low dose of prazosin caused no significant change in the affinity for [³H]prazosin or in the maximum number of α₁-adrenoceptor sites (B_max). However, treatment with prazosin (low dose) combined with stress increased the density of α₁-adrenoceptors with low affinity for prazosin. Treatment with a high dose of prazosin increased the density of α_1L-adrenoceptors, irrespective of stress loading. The densities of α_1A- and α_1B-adrenoceptors with high affinity for prazosin were increased only after treatment with a high dose of prazosin in combination with stress. These results indicate that three distinct α₁-adrenoceptor subtypes, α_1A, α_1B and α_1L, might be affected differently by treatment with prazosin and by stress.     

Postsynaptic α1-adrenoceptor mechanisms in rat vas deferens and ageing

Postsynaptic α₁-adrenoceptor mechanisms in vasa deferentia isolated from 3, 6, 18 and 40 week-old rats were studied by analysis of the concentration-response curve of noradrenaline and the Scatchard plot of specific binding of [³H]prazosin to microsomal fractions. The maximum tension developed by noradrenaline also increased with age from 3 to 18 weeks. The efficacy of noradrenaline and capacity of the maximum binding sites of [³H)prazosin increased with increasing age, while the dissociation constants of noradrenaline (K_A) and prazosin (K_d) were not changed with age. The increase of the maximum tension was proportional to the increase in efficacy. The increase of efficacy for noradrenaline in the vasa deferentia from rats of different ages is due to the increase in the total concentration of postsynaptic α₁-adrenoceptors.     

Further Evidence for the Existence of Two Forms of α2B-Adrenoceptors in Rat

Abstract: Analysis of saturation isotherms of the novel α₂-adrenoceptor antagonist radioligand [³H]-MK 912 revealed that the ligand labelled a homogenous population of α_2B-adrenoceptors in the neonatal rat lung with a K_d of 0.77 nM and a B_max of 231 fmol/mg protein. In rat kidney, combined saturation and competition experiments, using [³H]-MK 912 and the α_2A-adrenoceptor selective drug guanfacine, revealed that ~ 81% of the sites labelled by [³H]-MK 912 were α_2B-adrenoceptors and ~ 19% α_2A-adrenoceptors; the K_ds of [³H]-MK 912 being. 1.1 and 2.0 nM and the B_max 134 and 33 fmol/mg protein, respectively. The kidney α_2B-adrenoceptors were studied separately by using ~ 1.5 nM [³H]-MK 912 in the presence of 0.32 μM guanfacine, the latter which blocked ligand binding to α_2A-adrenoceptors completely. Analysis of drug competition curves obtained during these conditions revealed that 18 out of 20 different agonists and antagonists yielded steep and uniphasic competion curves which modelled best into one site fits. However, both guanoxabenz and LT 11 appeared to inhibit [³H]-MK 912 binding at two sites; the K_ds of guanoxabenz differing ~ 120-fold and that of LT 11 differing ~ 35-fold for the two sites. Moreover, the addition of mutual fixed concentrations of either 20 μM guanoxabenz or 20 μM LT 11 completely prevented the binding of the other compound to its high affinity site, indicating that both compounds labelled the same site with the high affinity. The analysis indicated that 29% of the sites were of high and 71% of low affinity. However, in the neonatal rat lung guanoxabenz and LT 11 (as well as 15 other compounds) yielded competition curves which modelled only into one site fits. The K_ds obtained in the lung correlated well with the K_ds obtained in the kidney for α_2B-adrenoceptors; for guanoxabenz and LT 11 the values from the lung were close to those determined in the kidney for the low affinity site for guanoxabenz and LT 11. Moreover, when the rat RNG α_2B-adrenoceptor was expressed in COS-7 cells and its binding properties tested using [³H]-MK 912 binding, guanoxabenz, LT 11 as well as a number of other drugs inhibited the ligand binding at a single α₂-adrenoceptor site; the drug K_ds being practically the same as those found for the neonatal rat lung. It is suggested that rat α_2B-adrenoceptors may exist in two forms: α_2B1 and α_2B2. The α_2B1-form, which shows high affinity for guanoxabenz and LT 11, is present in the adult rat kidney. The α_2B2-form, which shows low affinity for guanoxabenz and LT 11, is present in the neonatal rat lung as well as in the adult rat kidney and it is encoded by the RNG gene.     

Binding characteristics of [3H] guanfacine to rat brain α-adrenoceptors: Comparison with [3H]clonidine

The tritium-labeled α-adrenoceptor agonist and antihypertensive drug guanfacine, N-amidino-2-(2,6-dichlorophenyl)-acetamide (sp. act. 24.2 $\text{Ci}\text{mmole}$) was employed for a direct identification and characterization of α-adrenoceptors in rat brain membranes. Its usefulness as a radioligand was studied in comparison with [³H]clonidine (sp. act. 26.7 $\text{Ci}\text{mmole}$). The nonspecific binding of [³H]guanfacine to rat cerebral membranes was considerably more pronounced than that observed for [³H]clonidine. The specific binding of [³H] guanfacine (0.1–20 nM) and [³H]clonidine (0.1–20 nM) as defined as the excess over blanks containing (?)-norepinephrine (10μM) was saturable. Scatchard analyses of these binding data indicated single populations of binding sites for both ligands. K_D values of 3.9 ([³H]guanfacine) and 3.7 nM ([³H]clonidine) were calculated. Maximal number of specific binding sites amounted to 220 and 195 $\text{fmole}\text{mg}$ protein for [³H]guanfacine and [³H]clonidine, respectively. In case unlabeled guanfacine (1 μM) was used to characterize the specific binding of [³H] guanfacine, K_d value and maximal number of binding sites were about twice as high as determined in the presence of excess (?)-norepinephrine. The rate of association of both radioligands was rapid. Binding reached equilibrium by about 10–15 min of incubation. Half-maximal binding was attained at approximately 1–2 min. The rates of dissociation were biphasic. A rapid and a slow component were identified. The specific binding sites of [³H] guanfacine in rat brain possess the general characteristics of α₂-adrenoceptors. Selective antagonists of α₂-adrenoceptors, like yohimbine and rauwolscine strongly interfered with this binding. However, preferential blocking agents of α₁-adrenoceptors, such as prazosin and corynanthine, were weak competitors. The relative potency of agonists and antagonists in displacing [³H]guanfacine was identical to their effectiveness in competing for [³H]clonidine specific binding sites. It is concluded that [³H]guanfacine labels the same α₂-adrenoceptor population in rat brain as [³H]clonidine. However, [³H]guanfacine seems not as suitable as [³H]clonidine for routine use in the direct identification of α₂-adrenoceptors in view of its relatively high nonspecific binding.