Binding of adrenergic ligands ([3H]clonidine and [3H]WB-4101) to multiple sites in human brain

In order to identify alpha-adrenoceptors in post-mortem human brain and to detect the possible existence of multiple types of binding sites for adrenergic [³H]ligands, we studied the binding of [³H]clonidine and [³H]WB-4101 to human brain cerebral cortex, hippocampus, hypothalamus and striatum. Frontal cortex revealed two binding sites for [³H]clonidine (with K_D values of approximately 1 and 8 nM), as indicated by the biphasic Scatchard plot, the biphasic pattern of dissociation kinetics, and the biphasic inhibition by phentolamine on the binding of [³H]clonidine; the high-affinity site was heat-labile. Two high-affinity binding sites for [³H]WB-4101 were also detected in the human frontal cortex (with K_D values of about 0.09 and 1.5 nM), as revealed by a biphasic pattern of dissociation. A third site with low affinity binding for [³H]WB-4101 was detected by the biphasic inhibition by phentolamine (as well as by WB-4101 and prazosin) on the binding of [³H]WB-4101. The three other brain regions revealed very similar patterns exhibited by the frontal cortex, except that the density of the [³H]clonidine sites (of either high or low affinity) was highest in the hypothalamus, whereas the density of [³H]WB-4101 sites was highest in the hippocampus.     

Binding of [3H]quinuclidinyl benzilate to intestinal mucus: An artifact in identification of epithelial cell muscarinic receptors

The widely used muscarinic receptor ligand [³H]quinuclidinyl benzilate ([³H]QNB) was found to bind in a site-specific but artifactual manner to rat intestinal mucus, obscuring specific binding to muscarinic receptors on intestinal epithelial cells. Atropine inhibited [³H]QNB binding to mucus with an apparent IC₅₀ of 2.1 × 10^?7 M, compared to an IC₅₀ of 1.4 × 10^?8 M obtained with a homogenate of intestinal epithelial cells. Unlabeled QNB also inhibited binding of [³H]QNB to mucus but the apparent IC₅₀(4 × 10^?7 M) was about 300-fold greater than the IC₅₀ determined with a control tissue, heart muscle (IC₅₀, 1.2 × 10^?9M). [³H]QNB binding was saturable over the concentration range of 1–7 nM in the heart, with an apparent k_D of 0.76 nM. As expected from the high IC₅₀ for QNB in the mucus binding experiments, binding to mucus was not saturable over the 1–15 nM concentration range. Based on pH profiles and temperature dependency of binding, it seems unlikely that mucin, the primary component of mucus, was responsible for [³H]QNB binding to the mucus. The findings have implications for studies which involve binding of [³H]QNB in particular and other ligands in general to mucus-secreting epithelial tissues.     

Reduced number of α- and β-adrenergic receptors in the myocardium of rats exposed to tobacco smoke

The concentration of α- and β-adrenergic receptors-as measured by specific [³H]WB-4101 and (−)-[³H]-dihydroalprenolol binding-was diminished by 60% below control values in the hearts of hearts of rats exposed to tobacco smoke. These changes in receptor numbers took place almost immediately after tobacco smoke exposure and were rapidly reversible after termination of the exposure. The dissociation constant, K , for [³H]WB-4101 was identical in exposed (K_D = 0.34 ± 0.09 nM) and control (K_D = 0.35 ± 0.07 nM) hearts but was significantly different i in the case of (-)-[³H] dihydroalprenolol binding (exposed, (K_D = 2.83 ± 0.30 nM vs control K_D = 5.22 ± 0.61 nM). For β-receptor binding there was no significant difference between exposed and control animals in the K_i values (−)-epinephrine, (−)-norepinephrine, (−)-alprenolol, (±)-propranolol or timolol. (−)-Isoproterenol, however, was found to bind with lower affinity in exposed compared with control hearts. For α-receptor binding there was no significant difference between control and ‘smoked’ animals in the K_i values for (−)-epinephrine, (−)-norepinephrine or phentolamine. The decrease in α- and β-adrenergic receptor concentration may be related to the phenomenon of receptor desensitization resulting from a release of catecholamines in rats exposed to tobacco smoke.     

The influence of guanyl-5′-yl imidodiphosphate and sodium chloride on the binding of the muscarinic agonist, [3H] cis methyldioxolane

[³H]Quinuclydinyl benzylate([³H]QNB) binding was carried out on crude synaptosomal membranes isolated from cat cerebral cortex. The specific binding showed a single type of site with K_D 0.25 nM, Hill number 0.89 and B_max 114 pmol/g protein. The local anesthetics procaine, tetracaine, and the adrenergic antagonists phentolamine and propranolol, in concentrations between 1 nM and 500 μM, inhibited [³H]QNB binding with K_i varying between 9 μM for procaine and 80 μM for propranolol. The Hill coefficients obtained from logit/log plots suggested that there was no cooperativity between the binding sites for local anesthetics. At various concentrations the inhibition by procaine and propranolol may appear as competitive or non-competitive. The Hill numbers obtained from the saturation curves suggest that there was no cooperativity between anesthetics and [³H]QNB binding sites. Neither 1 mM Ca²⁺ nor Mg²⁺ affected [³H]QNB binding or the action of the drugs. The effect of local anesthetics and adrenergic antagonists was reversible and these drugs did not protect the muscarinic receptor from the deleterious effect of Triton X-100 as was the case with muscarinic agents. Our findings suggest that local anesthetics inhibit [³H]QNB binding to the muscarinic receptor by acting at some accessory site but not on the true receptor site. The possible mechanism of action of local anesthetics on synaptic transmission is discussed.     

Neuroleptic drug interactions with norepinephrine alpha receptor binding sites in rat brain.

Alpha adrenergic receptor sites in mammalian brain tissue can be labeled by the binding of [³H]WB-4101 (2-([2′,6′-dimethoxy] phenoxyethylamino) methyl benzodioxan), a potent α-adrenergic antagonist. Numerous neuroleptic drugs of phenothiazine, butyrophenone and thioxanthene classes are potent in competing for [³H]WB-4101 binding, with affinities resembling those of classic α-antagonists such as phentolamine and phenoxybenzamine. The potencies of neuroleptics in competing for WB-4101 binding sites correlate closely with their potencies in antagonizing norepinephrine and epinephrine induced lethality in rats, confirming that affinity for WB-4101 binding sites predicts α-receptor antagonism in vivo. The relative affinities of neuroleptics for WB-4101 binding sites and for dopamine receptors as labeled by [³H]haloperidol provides an index of the relative propensities of these drugs for eliciting autonomic side effects such as orthostatic hypotension and sedation.     

Multiple [3H]-nemonapride binding sites in calf brain

[³H]-Nemonapride has been the ligand of choice to label D₄ dopamine receptors. Its specificity was questioned when it was discovered that sigma (σ) sites were also labeled by [³H]-nemonapride. To further characterize the binding of [³H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [³H]-nemonapride labeled multiple sites. Dopaminergic and σ sites were the most prominent. The σ binding profile was σ-1 like with a K_i binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and σ sites, respectively, revealed additional, not previously characterized binding sites for [³H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [³H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met. Received: 19 September 1996 / Accepted: 12 March 1997     

Molecular size of [3H]WB-4101-binding sites in rat cortex as determined by radiation inactivation

The molecular weight of the [3H]WB-4101-binding sites in rat cerebral cortex was estimated by the irradiation-inactivation technique. The molecular weight was found to be dependent on the assay concentrations of the radioligand in the binding assay. Assays with a [3H]WB-4101 concentration of 0.25 nM showed a molecular weight of 62,100 daltons and 5.1 nM showed 50,800 daltons. Scatchard transformation of the [3H]WB-4101-binding data shows two binding sites (high-affinity: KD = 0.09 nM, Bmax = 9.1 pmoles/g; low-affinity: KD = 20 nM, Bmax = 80 pmoles/g). It is suggested that the two binding exist at two distinct molecules and in that case the observed molecular weights of 62,100 and 50,800 daltons are not true values because the determinations are carried out on a mixture of the two molecule populations. The distribution of the two binding sites was calculated for the two radioligand concentrations, 0.25 nM and 5.1 nM; and on this background the "true" molecular weights of the two [3H]WB-4101-binding sites were estimated to be 68,300 daltons for the high-affinity molecule and 41,400 daltons for the low-affinity molecule. Competition studies with a variety of adrenergic agonists and antagonists against [3H]WB-4101 supported the hypothesis that only the high-affinity binding site is an alpha-1-adrenoceptor.     

3H]Dihydroergocryptine binding to alpha-adrenergic receptors of human platelets. A reassessment using the selective radioligands [3H]prazosin, [3H]yohimbine, and [3H]rauwolscine

Which subtype(s) of the alpha-adrenergic receptor occurs on human platelets? Studies of platelet responsiveness to adrenergic compounds and indirect radioligand binding studies addressing this question have yielded contradictory conclusions. These binding studies employed the ligand [³H]dihydroergocryptine ([³H]DHE), an alpha-adrenergic antagonist that does not select between alpha₁- and alpha₂-adrenergic receptors and that also binds to other receptor types in some tissues. To determine the subtype of the platelet alpha-adrenergic receptor, we have examined the binding to intact human platelets of [³H]prazosin (alpha₁-selective), [³H]yohimbine (alpha₂-selective), and [³H]rauwolscine (alpha₂-selective), and we have compared the binding of these selective radioligands with that of [³H]DHE. [³H]Yohimbine and [³H]rauwolscine both bound with high affinity (K_D = 2.7 and 4.6 nM, respectively) to an equal number and a single class (Hill coefficient ～1.0) of sites (～300 per platelet), but [³H]yohimbine yielded lower nonspecific binding than did [³H]rauwolscine. In paired experiments, [³H]DHE bound to 1.5 times as many (phentolamine-displaceable) sites as did [³H]yohimibine or [³H]rauwolscine. Unlabeled vohimbine and epinephrine competed for fewer [³H]DHE binding sites than did phentolamine. Thus, in addition to binding to the alpha₂-adrenergic receptors identified by [³H]yohimbine and [³H]rauwolscine, [³H]DHE seems to bind to other sites on human platelets. The nature of these sites is not clear. We found that [³H]prazosin did not identify alpha₁-adrenergic receptors on platelets, and that phenoxybenzamine only inhibited [³H]yohimbine and [³H]DHE binding at higher concentrations than usually observed for alpha₁-adrenergic receptors. We conclude that (1) all alpha-adrenergic sites on human platelets are of the alpha₂ subtype, (2) [³H]DHE may bind to additional, as yet ill-defined, sites in addition to those sites identified by [³H]yohimbine and [³H]rauwolscine, and (3) [³H]yohimbine is the preferred antagonist radioligand for studying the alpha₂-adrenergic receptors on human platelets.     

BIOCHEMICAL CHARACTERISTICS OF MUSCARINIC CHOLINORECEPTORS IN SWINE TRACHEAL SMOOTH MUSCLE

• 1 The tritiated muscarinic cholinoreceptor antagonist quinuclidinyl benzilate, [³H]QNB, was used to characterize the muscarinic receptors associated with homogenized membrane of the smooth muscle from swine trachea. Based on receptor binding assays, the homogenate had specific, saturable, high-affinity receptors for [³H]QNB.
• 2 Specific binding was time- and temperature-dependent. The association of [³H]QNB with the muscarinic receptor reached equilibrium much sooner at 37°C than 25°C at a [³H]QNB concentration of 180 pM (30 min and 2 h, respectively). Equilibrium at both temperatures was attained within 5 min at a [³H]QNB concentration of 1800 pM. All remaining experiments were performed at 37°C.
• 3 Binding was saturable with respect to [³H]QNB and tissue concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (K_D) of 51±20 pM and a maximum receptor density (B_max) of 2.17±0.27 pmole/mg protein. The Hill coefficient for [³H]QNB binding was 1.07±0.16. The association (K₁) and dissociation (K_-1) rate constants were determined to be (5.51±0.16) × 10⁸ M^?1 min^?1 and (1.41±0.18) × 10^?2 min^?1, respectively. K_D calculated from the ratio of K₁ and K_-1 was 26.3±3.8 pM; this value is close to the value of K_D calculated from Scatchard plots of binding isotherms.
• 4 The density of muscarinic receptor binding sites was 10-fold greater in tracheal smooth muscle than in tracheal epithelium (0.20±0.03 pmole/mg protein). There is no difference between weanling and young adult swine in the density of muscarinic receptors in tracheal smooth muscle.
• 5 The nonselective muscarinic antagonists atropine, scopolamine and quinuclidinyl benzilate (QNB) competitively inhibited [³H]QNB binding to the homogenate with Hill coefficients of 0.9-1.0 and inhibition constants (K_i) of nanomolar range.
• 6 Competition with selective muscarinic antagonists pirenzepine and 3-quinuclidinyl xanthene-9-carboxylate (QNX) gave K_i values, 0.26 M and 0.78 nM, respectively, and Hill coefficients of approximately 1. There was a single population of [³H]QNB binding sites of the M₂ subtype for all tested muscarinic antagonists.
• 7 Competition with selective muscarinic agonists pilocarpine and carbachol yielded K_i values of micromolar range, Hill coefficients of less than 1, and revealed the existence of two binding sites (P < 0.01).

     

Reduced [H]quinuclidinyl benzilate binding to muscarinic receptors in disulfoton-tolerant mice

The organophosphate, acetylcholinesterase inhibitor, disulfoton, O,O-diethyl S-[2-(ethylthio)ethyl]phosphorodithioate, was given daily for 2 weeks to male mice at two different dosages. Clinical signs of poisoning disappeared in 5 days after the beginning of the treatment, i.e., the animals developed apparent tolerance to disulfoton toxicity. Tolerant mice were less sensitive to a lethal dose of carbachol and exhibited a decrease of [³H]quinuclidinyl benzilate ([³H]QNB) binding in forebrain, hindbrain, and ileum. Scatchard analysis of saturation experiments revealed a decrease in the density of receptors (B_max) in the disulfoton-treated mice, as compared with controls. No significant changes in affinity were found, except in the ileum. A time-course study showed a good parallelism between the decrease of [³H]QNB binding and the development of tolerance. Twenty-one days after the end of the disulfoton treatment AChE activity was still inhibited, but [³H]QNB binding had returned to normal levels. The recovery of [³H]QNB binding appears to be faster in ileum than in forebrain and hindbrain. These findings indicate that the development of tolerance to chronic organophosphate treatment is, at least partially, due to a reduction in the number of cholinergic receptors.     

Unusual binding of [3H] MDL 72222 in guinea pig hippocampus

[³H] MDL 72222 labeled a non-homogeneous population of sites in guinea pig hippocampal membranes (K_d1 = 1 nM; K_d2 = 60 nM). The binding was not sodium dependent. Competition studies with a variety of characterizing agents showed displacement of [³H] MDL 72222 binding by 5-HT uptake inhibitors. [³H] MDL 72222 binding was not effectively displaced by established 5-HT₃ antagonists. MDL 72222, fluoxetine, fluvoxamine and citalopram competitively inhibited the uptake of [³H] 5-HT into guinea pig hippocampal synaptosomes with K_i values of 1.97, 0.02, 0.023, 0.049 μM, respectively. The results demonstrate that [³H] MDL 72222 labels a non-homogeneous population of sites in guinea pig brain, as well as inhibiting 5-HT uptake into synaptosomal preparations.     

Human brain imidazoline receptors: further characterization with [3H]clonidine

The aim of the present study was to further characterize [³H]clonidine binding in the ventrolateral medulla of the human brainstem, the region involved in the vasodepressor effect of imidazoline drugs of the clonidine type. Under basal conditions, [³H]clonidine can bind both to the imidazoline receptors and to the α-adrenoceptors. The latter represent only a small part of the total [³H]clonidine binding with a B_max of 61 ± 13 fmol/mg proteins and a K_D of 4.9 ± 2.2 nM. Most of the binding was associated with imidazoline receptors with a K_D of 67 ± 13 nM and a B_max of 677 ± 136 fmol/mg protein. α-Adrenoceptor binding of [³H]clonidine could be completely prevented when membranes were either treated during preparation with the aIkylating agent phenoxybenzamine or incubated in the presence of 30 μM (−)-noradrenaline or in the presence of the non-hydrolysable analogue of GTP, guanylyl imidodiphosphate (Gpp(NH)p). When the α-adrenoceptors binding was prevented, we demonstrated the insensitivity of [³H]clonidine binding to Gpp(NH)p and showed that the competition between clonidine and idazoxan for imidazoline receptors was insensitive to Gpp(NH)p suggesting that imidazoline receptors are not G protein coupled receptors. The specificity of [³H]cloniding binding to imidazoline receptors in the human ventrolateral medulla indicates that these receptors are different from imidazole receptors as defined with p-aminoclonidine in the bovine brainstem.     

[3H]-Spiroperidol labels dopamine receptors in membranes from rabbit mesenteric artery

Summary The potent dopamine receptor antagonist [³H]-spiroperidol was used to label binding sites in a membrane fraction derived from rabbit mesenteric artery which had characteristics expected for dopamine receptors. The binding was of high affinity with an equilibrium dissociation constant (K_D) of 13.1 nM; it was saturable with 110 fmol of [³H]-spiroperidol bound/mg protein at maximal occupancy of the sites. Binding at 37° C was rapid and readily reversible with rate constants of 0.0154 nM^–1 min^–1 and 0.114 min^–1 for forward and reverse reaction, respectively. Dopamine receptor antagonists were about 100–200 times more potent than -adrenolytic drugs in competing for the [³H]-spiroperidol binding sites and dopamine was much more potent than (–)-noradrenaline, adrenaline, (–)-isoprenaline, clonidine or serotonin. It is concluded that in a membrane fraction of the rabbit mesenteric artery there exist binding sites for [³H]-spiroperidol indistinguishable from dopamine receptors. Thus the present results support the view that in vascular smooth muscle there exist specific dopamine receptors.This work was supported by the Deutsche Forschungsgemeinschaft     

Properties of the muscarinic cholinergic receptors in rat atrium

Summary 1. Properties of the muscarinic cholinergic receptor sites in the rat atrial homogenate and microsomal fraction were studied by the use of tritium labelled 3-quinuclidinyl benzilate ([³H]-QNB), a potent muscarinic antagonist. 2. The specific [³H]-QNB binding to the receptor sites displayed saturability, stereospecificity as well as reversibility. 3. The competition studies showed that muscarinic antagonists were more potent than muscarinic agonists. 4. Certain neuromuscular blocking agents, antipsychotics, antiarrhythmics and antihistamines also were capable of interacting with the [³H]-QNB binding sites. However, - and -adrenergic agents, calcium antagonist (D-600) and ionophore (A-23187) failed to show any effect. 5. Analyses by the double reciprocal plot, Hill plot and Scatchard plot of the dependence of the specific [³H]-QNB binding on the concentration of QNB suggested that binding was occurring to a single population of receptor sites in the atrial homogenate or microsomal fraction. Further, there was no evidence of any detectable site to site interactions (positive or negative cooperative type). From the Scatchard plot, the equilibrium dissociation constant (K_D) of 1.1 nM was calculated and the Hill coefficient was close to 1.0 6. Interaction of the muscarinic antagonists with the [³H]-QNB sites showed the Hill coefficients close to 1.0 whereas for the agonists, the coefficients were much less than 1.0 indicating that agonist-receptor site interactions have some different characteristics from those following antagonist-receptor site interaction. 7. The rate and the maximal level of QNB binding to the receptor sites was markedly influenced by the temperature; various cations, on the other hand, displayed no effect either on the association or dissociation of QNB binding. The specific QNB binding exhibited a broad pH optimum from pH 6.0–8.5. 8. Treatment of the membrane fraction (or homogenate) with either phospholipase A or C and with p-chloromercuribenzoate caused significant inhibition of [³H]-QNB binding. 9. The QNB binding site was resistant to tryptic digestion. Even when about 40% of the membrane proteins were removed by the tryptic proteolysis, the [³H]-QNB binding ability of the membrane remained unaffected; in fact, the removal of tryptic proteolytic products by centrifugation markedly increased the specific QNB binding to the membrane.     

[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.     

Inhibition of [3H]norepinephrine uptake in peripheral organs of some mammalian species by ouabain

In order to demonstrate the possible involvement of (Na⁺ + K⁺)-ATPase in the high affinity uptake of [³H]-norepinephrine in the sympathetic nerve endings, the effect of ouabain on [³H]norepinephrine uptake in spleen and heart slices of five mammalian species was examined. The ouabain sensitivity of [³H]norepinehrine uptake in the heart slices from various species, as determined by the estimation of IC₅₀, was, in increasing order, lamb (2.3 μM) < calf (2.5 μM) < guinea pig (4 μM) < rabbit (10 μM) << rat (>500 μM). The IC₅₀ values in the spleen slices were: lamb (1 μM) < calf (3.2 μM) < rabbit (9.5 μM) < guinea pig (25 μM) << rat (>500μM). The IC₅₀ values for the inhibition of specific [³H]ouabain binding in the microsomal fractions of spleen and heart of the five mammalian species by ouabain were similar to the IC₅₀ values for the inhibition of [³H]norepinephrine uptake by the cardiac glycoside. Since ouabain is known to bind exclusively to (Na⁺ + K⁺)-ATPase of a microsomal fraction, these results suggest that the inhibition of [³H]norepinephrine uptake in the sympathetic nerve endings by ouabain is mediated by the inhibition of (Na⁺ + K⁺)-ATPase.     

Binding of an AMPA receptor potentiator ([H]LY395153) to native and recombinant AMPA receptors

LY395153 is a member of a newly described class of arylpropylsulfonamide AMPA receptor potentiators. Here, we characterize and compare [³H]LY395153 binding to native AMPA receptors from rat cerebral cortex and recombinant human GluR4_flip receptors expressed in HEK293 cells. -Glutamate and AMPA increased [³H]LY395153 binding to both native and recombinant AMPA receptors in a concentration dependent and stereoselective manner; this effect of AMPA receptor agonists reflects an apparent increase in ligand affinity. In the presence of -glutamate (500 μM), [³H]LY395153 binding is saturable; the affinity of this radioligand is slightly, albeit statistically significantly higher at human GluR4_flip (K_d=55.6±5.3 nM) than rat cortical receptors (K_d=110±15.1 nM). NBQX competitively inhibited -glutamate-induced increases in [³H]LY395153 binding in both native and recombinant receptors, whilst LY303070 (the active isomer of GYKI53655) noncompetitively inhibited this effect in native, but not recombinant receptors. The prototypic AMPA receptor potentiator cyclothiazide competitively inhibited [³H]LY395153 binding with a potency (K_i7 μM) comparable to EC₅₀ values reported in electrophysiological studies. In contrast, the structurally unrelated AMPA receptor potentiator CX 516 did not inhibit [³H]LY395153 binding at concentrations of up to 600 μM. Further, at concentrations reported to facilitate AMPA receptor desensitization, thiocyanate acts as a competitive inhibitor of [³H]LY395153 binding. [³H]LY395153 binding was unaffected by a variety of structurally (and mechanistically) diverse compounds tested at a concentration of 10 μM. These data indicate [³H]LY395153 is a useful probe for labeling a unique modulatory site on both native and recombinant AMPA receptors.     

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

Summary The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [³H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [³H]QNB was saturable, reversible and of high affinity (K _D = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M₁), AF-DX 116 (M₂) and 4-DAMP (M₃). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M₃ receptors and a much smaller population (12%) exhibiting characteristics of M₂ receptors. The binding curve for the displacement of [³H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (K _D = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (K _D = 4.4 M). When oxotremorine displacement of [³H]QNB binding was determined in the presence GTPS, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [³²P]NAD caused the [³²P]-labelling of a 40 kD protein that may represent the subunit(s) of G proteins that are known to be NAD-ribosylated by the toxin. We conclude that both M₃ and M₂ receptors may be coupled to G proteins in a pertussis-sensitive manner. Send offprint requests to T. W. Honeyman at the above address     

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.     

Properties of the binding sites of [3H]9-methyl-7-bromoeudistomin D in bovine aortic smooth muscle microsomes

Abstract— [³H]9-Methyl-7-bromoeudistomin D ([³H]MBED), a powerful caffeine-like Ca²⁺ releaser, binds to the caffeine binding site of terminal cisternae of skeletal muscle sarcoplasmic reticulum and activates Ca²⁺-induced Ca²⁺ release. Properties of the binding site of [³H]MBED were investigated in aortic smooth muscle. The specific activity was higher in microsomes than in other fractions. [³H]MBED binding sites in smooth muscle microsomes were of a single class with a high affinity (K_D 50 Nm ), comparable with that in skeletal muscle sarcoplasmic reticulum. Caffeine competitively inhibited [³H]MBED binding, indicating MBED shares the same binding site with caffeine. Solubilization and fractionation of the microsomes gave two fractions of [³H]MBED binding activities. These results suggest that, in smooth muscle, there are multiple binding sites of [³H]MBED and caffeine, which might correspond to different pharmacological actions of caffeine on smooth muscle. Therefore, [³H]MBED, which binds to the different binding sites of caffeine, is useful as a probe for investigation of the actions of caffeine at the molecular level.