Receptor Binding Profiles of Amiloride Analogues Provide no Evidence for a Link Between Receptors and the Na+/H+ Exchange,But Indicate a Common Structure on Receptor Proteins

Abstract

Amiloride and its analogues affect radioligand binding to the adenosine-A₁ receptor. In this paper, the specificity of this effect is investigated by generating receptor binding profiles for amiloride and two of its analogues. A limited structure-activity relationships study is performed to probe the relationship between inhibition of receptor binding by amiloride analogues and the effects of these compounds on Na⁺ transport, in particular Na⁺/H⁺ exchange. The receptor binding profiles of amiloride, benzamil and 5′-(N,N-hexamethylene)amiloride (HMA) indicate that the compounds affect a variety of receptors and that none of the compounds is highly selective for any of these. The SAR study indicates that it is very unlikely that a direct coupling between receptors and Na⁺/H⁺ exchange or another amiloride-sensitive ion transport system is responsible for the inhibition of receptor binding. A correlation between the signal transduction systems coupled to the receptors involved and the potency of the amiloride analogues is also absent. The varying nature of the receptors, affected by amiloride or its analogues, suggests a wide-spread presence of an amiloride binding site on receptors and other membrane proteins.     

Amiloride is a cholinergic antagonist in the rabbit pancreas

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na⁺), or in a medium containing only 25 mM Na⁺. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin (PzO) is not affected. Amiloride also inhibits the carbachol-induced ⁴⁵Ca efflux from rabbit pancreatic acini, but again not that induced by PzO. The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and ⁴⁵Ca efflux are 40 and 80 μM, respectively. Amiloride also competitively inhibits the specific binding of [³H]quinuclidinyl benzylate ([³H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

The mode of interaction of amiloride and some of its analogues with the adenosine A1 receptor.

Amiloride, a potassium sparing diuretic, inhibits adenosine A1 receptor-radioligand binding in calf and rat brain membranes in the low micromolar range. The drug interacted with the A1 receptor in a manner different from classical A1 ligands, but structure-activity relationship studies indicated that this inhibitory effect is not related to the ion transport inhibiting properties of amiloride (Garritsen et al., 1990a,b) In the present study, the question is addressed how amiloride interacts with the adenosine A1 receptor. Amiloride and two of its analogues, in concentrations equivalent to their Ki values in displacement studies, decrease the affinity of the A1 antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine, but not the maximal binding capacity of the radioligand. Furthermore, the dissociation rate of the receptor-ligand complex is unaltered in the presence of amiloride or its analogues in a concentration exceeding the Ki value 10-fold. These characteristics argue for a purely competitive mode of interaction. The functional consequences of the interaction between amiloride analogues and the A1 receptor were investigated at the level of cyclic adenosine 3',5'-monophosphate (cAMP) formation. The amiloride analogue 5-(N-butyl-N-methyl) amiloride (MBA) reversed A1-receptor mediated inhibition of forskolin-stimulated cAMP formation in rat fat cell membranes. In this model, the antagonist potency of MBA is ca 5 microM. This value is in fair agreement with a Ki value of 3.5 microM in binding assays under similar conditions. In conclusion, amiloride inhibits A1 receptor binding in an apparently competitive manner. This suggests that the binding sites of amiloride and the classic A1 receptor ligands may at least partially overlap.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of porcine brain alpha 2-adrenergic receptors by Na+,H+ and inhibitors of Na+/H+ exchange

Previous reports from this laboratory have demonstrated that alpha 2-adrenergic receptors accelerate Na+/H+ exchange in NG108-15 neuroblastoma X glioma cells and evoke platelet secretion via a pathway involving Na+/H+ exchange. The present studies were designed to examine whether agents that interact with Na+/H+ antiporters also might influence alpha 2-adrenergic receptor-ligand interactions. We observed that Na+ decreases receptor affinity for the agonists epinephrine, norepinephrine, and UK14304 and slightly increases receptor affinity for the antagonists yohimbine and idazoxan in digitonin-solubilized preparations from porcine brain cortex. Increases in [H+] also decrease receptor affinity for agonists and cause either a slight increase or no change in receptor affinity for antagonists. Amiloride analogs accelerate the rate of [3H] yohimbine dissociation from digitonin-solubilized receptors with a relative effectiveness that parallels their ability to block Na+/H+ exchange in other systems. Interestingly, these modulatory effects of Na+,H+ and 5-amino-substituted analogs of amiloride are retained in homogeneous preparations of the alpha 2-adrenergic receptor, suggesting that the allosteric-binding sites for these agents are on the receptor-binding protein itself.     

Differentiating Adenosine Receptors and Adenosine Uptake Sites in Brain

Abstract

The characteristics of adenosine receptors and adenosine uptake sites in brain are presented. High affinity adenosine receptors of the A₁ type bind [³H]cyclohexyladenosine ([³H]CHA) and [³ H]diethyl-phenyl-xanthine ([³H]DPX) with 10^?9 potency while adenosine uptake sites are labeled 10^?10 potency with [³ H]nitrobenzyl-thioinosine ([³H]NBI). NBI does not inhibit either [³H]CHA (agonist) or [³H]DPX (antagonist) binding to adenosine receptors in brain cortical membranes and conversely CHA and other adenosine receptor ligands are very poor inhibitors of [³H]NBI binding to adenosine uptake sites. A number of other differences between the receptor and uptake site are discussed which provide rather strong evidence that these two sites are quite distinct and that the labeled ligands used represent specific probes for each site.     

Alpha1 Adrenergic Receptors in the Porcine Pituitary Neurointermediate Lobe: Detection with [3H]Ketanserin

Abstract

[³H]Ketanserin, a serotonin receptor antagonist, labelled high affinity, saturable sites in homogenates of porcine neurointermediate lobe tissue. Cinanserin, a potent and selective serotonin receptor antagonist, inhibited the specific binding of 5 × 10^-10M [³H]ketanserin with a high affinity component representing 20% of the total binding. Prazosin, a potent and selective alpha₁ adrenergic antagonist, inhibited [³H]ketanserin binding with a high affinity component representing 60% of total binding. The prazosin-specific component was demonstrated to be distinct from the cinanserin-specific component. 10^-7M cinanserin was co-incubated with [³H]ketanserin to eliminate the serotonergic component of the binding and allow pharmacological characterization of the remaining prazosin-specific component. The prazosin-specific binding of [³H]ketanserin binding closely resembled the results of experiments using [³H]prazosin to label alpha₁ receptors in neurointermediate lobe tissue homogenates. Ketanserin was found to be sevenfold more potent in inhibiting [³H]prazosin binding to alpha₁ adrenergic receptors in the neurointermediate lobe tissue than in brain tissue. This observation explains why low concentrations of [³H]ketanserin can selectively label serotonin receptors in the brain but will label both adrenergic and serotonin receptors in the neurointermediate lobe.     

[3H]-BIBP3226 and [3H]-NPY Binding to Intact SK-N-MC Cells and CHO Cells Expressing the Human Y1 Receptor

Abstract

We have studied the binding of [³H]-NPY and the newly developed non-peptide Y₁ receptor antagonist [³H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y₁ receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [³H]-NPY binding was slow, the binding kinetics of [³H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y₁ agonists NPY, PYY and [Leu³¹-Pro³⁴]-NPY completely and potently displaced [³H]-NPY binding, they could only displace 70 to 80 % of the [³H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [³H]-NPY binding sites in CHO-Y1 cells whereas [³H]-BIBP3226 binding parameters remained unchanged.     

Interaction of amiloride with alpha-adrenoreceptors: evidence from radioligand binding studies

We investigated the effect of amiloride on alpha-adrenoreceptors (alpha 1 and alpha 2) using radioligand binding techniques. Amiloride inhibited [3H]yohimbine and [3H]prazosin binding to alpha 2- and alpha 1-adrenoreceptors, respectively, from various tissues in a concentration-dependent manner. Amiloride was approximately 9-12 times more potent in inhibiting [3H]yohimbine binding to alpha 2-adrenoreceptors from rat tissues than from other mammalian tissues. However, it had almost the same potency in inhibiting [3H]prazosin binding to alpha 1-adrenoreceptors from rat as well as other mammalian tissues. Further, in rat tissues, amiloride was approximately 10 times more potent in inhibiting [3H]yohimbine than [3H]prazosin binding. Amiloride inhibited [3H]yohimbine binding noncompetitively and [3H]prazosin binding competitively. The inhibition of [3H]yohimbine and [3H]prazosin binding by amiloride was reversible. Since amiloride has been shown to be an inhibitor of Na+-H+ exchanger protein, we believe that it regulates the alpha 2-adrenoreceptors by binding to Na+ -H+ exchanger protein. Triamterene, a compound similar to amiloride in regard to diuretic effect, had very little effect on [3H]yohimbine and [3H]prazosin binding to rat kidney membranes, suggesting that the alpha-adrenoreceptor antagonistic properties of amiloride are not related to its antikaliuretic effect. The results of the present study suggest that some of the pharmacological actions of amiloride (antihypertensive and diuretic effects) can be explained in part by its regulatory effect on both alpha 1- and alpha 2-adrenoreceptors.     

Inhibition and labeling of the rat renal Na+/H+-exchanger by an antagonist of muscarinic acetylcholine receptors

A covalently binding label for muscarinic acetylcholine receptors, propylbenzilylcholine mustard (PrBCM), irreversibly inhibits the Na+/H+ exchanger in rat renal brush-border membrane vesicles. Substrates of the antiporter, Na+ and Li+, as well as inhibitors, amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and propranolol, protect the antiporter from inactivation by PrBCM. With [3H]PrBCM a band with an app. Mr of 65 kDa is predominantly labeled. Amiloride protects this band from labeling with [3H]PrBCM and [14C]-N,N'-dicyclohexylcarbodiimide (DCCD) proving its identity with the renal Na+/H+ exchanger. Our data reveal a specific interaction of PrBCM with the Na+/H+ exchanger and suggest structural relations between antiporter and receptors.     

Increased lung uptake of liposomes coated with polysaccharides

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na+), or in a medium containing only 25 mM Na+. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin ( PzO ) is not affected. Amiloride also inhibits the carbachol-induced 45Ca efflux from rabbit pancreatic acini, but again not that induced by PzO . The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and 45Ca efflux are 40 and 80 microM, respectively. Amiloride also competitively inhibits the specific binding of [3H]quinuclidinyl benzylate ( [3H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Autoradiographic Visualization of Kappa Opioid Receptors with Labelled Dynorphins in Guinea Pig Brain

Abstract

The distribution of kappa opioid receptors in guinea pig brain was measured by in vitro receptor autoradiography using [³H]dynorphin A_1–9, [³H]dynorphin A_1–8 and [³H]bremazocine as ligands. The sites labelled by the two dynorphins had identical, heterogeneous distributions in brain sections. High levels of kappa receptors were seen in striatum, claustrum, nucleus accumbens and laminae V and VI of the cerebral cortex. The substantia nigra and superior colliculus also had high dynorphin binding levels. The [³H]dynorphin autoradiographs were closely similar to those obtained using [³H]bremazocine in the presence of mu and delta receptor displacers. It is concluded that tritiated dynorphin A fragments can be used for autoradiographic studies of kappa opioid receptors in brain.     

Amiloride potentiates atrial natriuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa

The interaction of atrial natriuretic factor (ANF) with the diuretic amiloride was studied in bovine adrenal zona glomerulosa. Amiloride enhances 2 to 3-fold high affinity binding of [125I] ANF to zona glomerulosa membrane receptor with an ED50 of 10 microM. This effect is due to a recruitement of high affinity receptor sites and to an increase of their affinity from a Kd of 23 to 8 pM. This enhancing effect is almost equipotently elicited by guanabenz, while clonidine is 20-fold less potent and arginine is inactive. ATP reduces by 30 to 50% [125I] ANF binding with an IC50 of 50 microM. Amiloride and ATP opposite effects on [125I] ANF binding are mutually competitive. Low concentrations of amiloride (less than 100 microM) potentiate the inhibitory effect of ANF in hormone-stimulated steroid secretion with a 3-fold decrease in ANF IC50 at 10 microM amiloride. Higher concentrations of amiloride (greater than 100 microM) directly inhibit aldosterone secretion with an IC50 of 500 microM and a maximum of 80 to 100% reversal of stimulation by various secretagogues. These results indicate that amiloride synergistically potentiates ANF inhibitory action by altering ANF receptor binding properties. They also suggest a role for sodium transport and for phosphorylation-dephosphorylation mechanisms in the mode of action of ANF.     

[3H]5,7-Dichlorokynurenic Acid Recognizes two Binding Sites in Rat Cerebral Cortex Membranes

Abstract

Binding of [³H]5,7-dichlorokynurenic acid ([³H]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor channel complex, was characterized in synaptic plasma membranes from rat cerebral cortex. Non linear curve fitting of [³H]DCKA saturation and homologous displacement isotherms indicated the existence of two binding sites: a specific, saturable, high affinity site, with a pK_D value of 7.24 (K_D = 57.5 nmol/1) and a maximum binding value (B_max) of 6.9 pmol/mg of protein and a second site, with micromolar affinity. The pharmacological profile of both binding components was determined by studying the effect on [³H]DCKA and [³H]glycine binding of a series of compounds known to interact with different excitatory and inhibitory amino acid receptors. These studies confirmed the identity of the high affinity site of [³H]DCKA binding with the strychnine-insensitive glycine site of the NMDA receptor channel complex. 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV 150526A), a new, high affinity, selective glycine site antagonist (1), was the most potent inhibitor of this component of binding (pK_i = 8.24, K_i = 5.6 nmol/1). The low affinity component of [³H]DCKA binding was insensitive to the agonists glycine and D-serine and the partial agonist (±)-3-amino-1-hydroxy-2-pyrrolidone (HA 966), though recognised by glycine site antagonists. The precise nature of this second, low affinity [³H]DCKA binding site remains to be elucidated.     

[3H] spiroperidol binding to a putative dopaminergic receptor in rat pituitary gland

A class of high affinity DA receptor sites has been identified with [³H] SPIR in the anterior and pisterior lobes of rat pituitary gland. Competitive studies with DA receptor antagonists and agonists clearly demonstrated that [³H] SPIR stereospecifically labels a true dopaminergic receptor in both lobes. The physiological significance of these receptors, although still unclear, may be that of controlling the secretion of pituitary hormones and peptides.     

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes

Abstract

The binding characteristics of radiolabeled N⁶-(cyclohexyl)adenosine ([³H]CHA), N⁶-(R-phenylisopropyl)adenosine ([³H]R-PIA), 5′-N-ethylcarboxamidoadenosine ([³H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5′-N-ethylcarboxamidoadenosine ([³H]CGS 21680), to rat testis membranes were investigated. Specific binding of [³H]CGS 21680, a selective agonist for the A_2a adenosine receptor, was very modest whilst the nonselective agonist [³H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [³H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the At adenosine receptor, [³H]CHA and [³H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A₁ adenosine receptor with a potency order for agonists of CHA R-PIA > NECA > N⁶-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A₃ adenosine receptor in these membranes we selectively blocked the A₁ receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [³H]CHA and [³H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A₃ adenosine receptor.     

Characterization of the Binding of a Novel Non-Xanthine Adenosine Antagonist Radioligand, [3H]CGS 15943, to Multiple Affinity States of the Adenosine A1 Receptor in the Rat Cortex

Abstract

The use of xanthine adenosine receptor antagonists such as 1,3-dipropyl-8-phenylxanthine (DPX) as radioligands for the characterization of adenosine receptor Pharmacology have been limited by their high lipophilicity, low specific activity, and their general lack of selectivity and affinity for adenosine receptors. Recent attempts to address the technical problems associated with this class of compounds has resulted in the development of several xanthine derivatives (e.g. the functionalized xanthine congeners [³H]XCC and [³H]XAC², and [³H]CPX³) which bind with high and selective affinity to the adenosine A₁ receptor subtype. Based on efforts to optimize non-xanthine adenosine receptor antagonists, CGS 15943, a derivative of the pyrazoloquinazoline benzodiazepine receptor inverse agonist CGS 8216⁵, represents the first reported non-xanthine structure that potently blocks adenosine receptors⁶. CGS 15943 has nanomolar affinity for both A₁ and A₂ receptor subtypes⁶. However, in contrast to many of the xanthine adenosine receptor antagonists, CGS 15943 is not a phosphodiesterase inhibitor and does not interact with adenosine transporter sites⁶. This compound is a potent and selective adenosine receptor antagonist in vivo ⁷ with a solubility/affinity ratio of greater than 1000⁷. In the present studies, the binding of [³H]CGS 15943 to the adenosine A₁ receptor was characterized.     

Guanylpirenzepine Distinguishes Between Neuronal m1 and m4 Muscarinic Receptor Subtypes

Abstract

Guanylpirenzepine, a polar, non-quaternary analog of pirenzepine, exhibited a novel binding behavior in rat brain regions: in competition binding experiments against [³H]pirenzepine labeling the M₁ receptor in membranes from cerebral cortex, hippocampus and striatum, the compound, differently from pirenzepine, displayed heterogeneous binding curves. Computer assisted analysis of these curves, evidenced the existence of two populations of binding sites: a large proportion (84–89%) of high affinity receptors (K_H = 64–92 nM) and a remainder with very low affinity (K_L = 19–28 μM). Like pirenzepine, quanylpirenzepine showed low affinity for the glandular M₃ and the cardiac M₂ receptors when [³H]N-methylscopolamine was used to label the receptors in membranes from these two tissues; affinity values for guanylpirenzepine were 1336 and 5790 nM respectively, vs 323 and 683 nM for pirenzepine. We conclude that guanylpirenzepine is able to discriminate between m1 and m4 receptor subtypes and may represent a new tool for deeper studies on mascarinic receptors classification.     

Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [³H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [³H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors.Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [³H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [³H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [³H]AMP for renal receptors. The cyclic [³H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [³H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [³H]AMP was enhanced by ATP or AMP.Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N⁶-butyryl-adenosine cyclic 3′,5′-monophosphate or N⁶,O²-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [³H]AMP in competitive binding assays.This study suggested that the membrane cyclic [³H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.     

Modulation of alpha-1 adrenergic receptors in rat brain following chronic reserpine

Functional denervation of the central adrenergic receptors by 30 daily injections of reserpine (0.25 mg/kg/day s.c.) produced an increase in the B_max of alpha-l adrenergic receptor binding sites labeled by [³H]prazosin. A similar increase was also observed for the alpha-1 adrenergic receptor component of [³H]WB4101 binding in the hippocampus but not in the cortex. No change in the lower affinity [³H]WB4101 binding site, which identifies S-l serotonin receptors was detected after this treatment. These data support the hypothesis that alpha-1 receptors are regulated by their neurotransmitter and may explain why previous studies have not detected alpha-1 receptor increases following 6-hydroxydopamine lesions of the dorsal bundle and locus coeruleus.     

Characterization of Muscarinic Acetylcholine Receptors in Cultured Bovine Aortic Endothelial Cells

Abstract

The binding characteristics of [³H]quinuclidinyl benzilate ([³H]QNB) to isolated crude membranes of cultured bovine aortic endothelial cells were investigated. [³H]QNB bound to endothelial cell membranes with high affinity (k_D = 0.056 nM) and limited capacity (132 fmol/mg DNA). The binding specificity, order of affinity and inhibition constants (K_i) were determined by displacement of bound [³H]QNB with unlabeled ligands. The order of affinity was QNB > atropine > 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) > p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) (M₃ antagonist) > pirenzepine (M₁ antagonist) > AFDX-116 (M₂ antagonist) > (4-hydroxy-2-butynyl) trimethylammonium chloride m-chlorocarbanilate (McN-A-343, M₁ agonist). These observations suggest that muscarinic receptors of endothelial cells in culture are likely to be of M₃ and M₁ subtype. Northern blot analysis of receptor subtypes using cDNA probes did not provide conclusive results due to the low level expression of these receptors in cultured cells. Solubilization of protein bound [³H]QNB with 1% digitonin and 0.02% cholate followed by analysis on sucrose density gradients demonstrated the presence of a specifically bound [³H]QNB-protein complex sedimenting at the 6.2S region of the gradient. These data demonstrate the presence of muscarinic acetylcholine receptor protein in cultured bovine aortic endothelial cells.