α-Adrenoceptor assays.

α-Adrenoceptors mediate responses to activation of both peripheral sympathetic nerves and central noradrenergic neurons. They also serve as autoreceptors that modulate the release of norepinephrine (NE) and other neurotransmitters. There are two major classes of α-adrenoceptors, the α(1)- and α(2). Each class is subdivided into three subtypes: α(1A), α(1B), α(1D), and α(2A), α(2B), α(2C). Described in this unit are in vitro isolated tissue methods used to study α-adrenoceptor functions and to identify novel ligands for these receptors. Detailed protocols describing use of isolated tissues to study the various α(1)- and α(2)-adrenoceptor subtypes are provided.

Characterization of postsynaptic alpha-adrenoceptors in the arteries supplying the oviduct.

1. In vitro experiments were designed to characterize postjunctional alpha-adrenoceptor subtypes in ring segments (1 mm length; outer diameter 300-500 microns) from arteries supplying the oviduct of the heifer. 2. Noradrenaline, adrenaline and phenylephrine evoked concentration-dependent contractile responses. The pD2 values were 5.67, 5.89 and 5.93, respectively. Medetomidine clonidine and B-HT 920 (2-amino-6-allyl-5,6,7,8-tetra-hydro-4H-(thiazo)-4,5-d-azepoine ) were ineffective. 3. The alpha-adrenoceptor selective antagonists, prazosin (1 nM-0.1 microM) and rauwolscine (0.1-10 microM) competitively antagonized the response to noradrenaline. The pA2 values were 9.38 and 6.83, respectively. 4. The dissociation constant (KD) for noradrenaline calculated by use of the irreversible antagonist, dibenamine, was 3.95 (2.09-5.81) microM. The occupancy-response relationship was non-linear. Half-maximal response to noradrenaline was obtained with 22% receptor occupancy while maximal response required 100% occupancy. 5. B-HT 920 evoked a biphasic contractile concentration-dependent response in preparations incubated in a physiological solution containing 20 mM K+, 0.1 microM prazosin and 1 microM propranolol. Rauwolscine 0.1 microM significantly (P less than 0.01) blocked the first component of the B-HT 920 concentration-response curve with an apparent pA2 value of 8.52 (7.86-9.18). 6. These results strongly suggest that alpha-adrenoceptors in oviductal arteries are mainly of the alpha 1 subtype, although a possible role for alpha 2-adrenoceptors cannot be excluded.

Synthesis of a yohimbine-agarose matrix useful for large-scale and micropurification of multiple alpha 2-receptor subtypes.

We have provided a detailed protocol for the synthesis of a yohimbine-agarose matrix that has been shown to be effective for isolation of the alpha 2A-adrenergic receptor from human platelet and purification of the alpha 2A-adrenergic receptor to apparent homogeneity from porcine brain cortex using chromatography on only two sequential yohimbine-agarose columns. In addition, this affinity matrix also interacts with alpha 2 receptors of the alpha 2B subtype extracted from cultured NG108-15 cells. Finally, this affinity matrix has proven useful for monitoring posttranslational modifications of the receptor in digitonin extracts of metabolically labeled cells. Thus, this affinity matrix can be exploited for the purification of multiple alpha 2-adrenergic receptor subtypes on both a macro- and microscale and should be of value to any laboratory exploring the molecular basis for alpha 2-adrenergic functions.

A comparison of the effects of angiotensin II and Bay K 8644 on responses to noradrenaline mediated via postjunctional alpha 1-and alpha 2-adrenoceptors in rabbit isolated blood vessels.

1. The effects of angiotensin II (AII) and Bay K 8644 on responses to noradrenaline (NA) mediated via postjunctional alpha 1- and/or alpha 2-adrenoceptors have been compared in three isolated venous preparations from the rabbit, the lateral saphenous vein, the left renal vein and the ear vein. 2. A similar action of AII and Bay K 8644 was observed only in the lateral saphenous vein; each potentiated responses to NA after isolation of a homogeneous population of postjunctional alpha 2- adrenoceptors. However, even in this preparation the mechanism of action for these agents was not identical. The sensitivity of KCl-induced contraction to changes in extracellular calcium ions (reflecting activation of voltage-dependent Ca2+ channels) was enhanced by Bay K 8644 but reduced by AII. 3. All produced a selective facilitation of responses mediated via postjunctional alpha 2-adrenoceptors. In the lateral saphenous vein it reduced the effectiveness of prazosin and facilitated responses after isolation of alpha 2-adrenoceptors with phenoxybenzamine and rauwolscine. It directly enhanced responses to NA in the ear vein, where only alpha 2-adrenoceptors are involved. In contrast, AII did not influence responses mediated via postjunctional alpha 1-adrenoceptors in the left renal vein (even after the receptor reserve had been removed with phenoxybenzamine) nor the 'rauwolscine-resistant' component of responses to NA in the saphenous vein. 4. Bay K 8644 enhanced contractile responses to NA mediated both via alpha 2-adrenoceptors, in the lateral saphenous vein, and via alpha 1-adrenoceptors in the left renal vein. Thus, unlike angiotensin II, no preferential effect was apparent. 5. Bay K 8644 was inactive against responses to NA in the rabbit isolated ear vein. Since the sustained component of responses to NA in this preparation is dependent upon the influx of extracellular Ca2 , these observations suggest that the influx of Ca2+ stimulated by NA is mediated via receptor-operated (1,4-dihydropyridine-resistant) Ca2 + channels.

Isolation of rat genomic clones encoding subtypes of the alpha 2-adrenergic receptor. Identification of a unique receptor subtype.

alpha 2-Adrenergic receptors (alpha 2-AR) exist as subtypes that are expressed in a tissue-specific manner and differ in 1) their ligand recognition properties, 2) their extent of receptor protein glycosylation, and possible 3) their mechanism of signal transduction. Genomic or cDNA clones encoding three receptor subtypes have been characterized; however, both functional and radioligand binding studies in rodents suggest the existence of a fourth receptor subtype. To isolate the rat genes encoding receptor subtypes we screened a rat genomic library with an oligonucleotide probe encompassing the third membrane span of the human C-4 alpha 2-AR. Two intronless rat genes were isolated that encode distinct receptor subtypes (RG10, RG20). RG10 and RG20 encode proteins of 458 and 450 amino acids, respectively, that are 56% homologous and possess the structural features expected of this class of membrane-bound receptors. RG10 identifies a mRNA species of approximately 2500 nucleotides that is found primarily in brain, whereas RG20 identifies a larger mRNA species (approximately 4000 nucleotides) that is found in several tissues including brain, kidney, and salivary gland. RG10 is 88% homologous to the human C-4 alpha 2-AR and exhibits similar binding properties ( [3H]rauwolscine KD = 0.7 +/- 0.3 nM) as determined following transient expression of the receptor in COS-1 cells. RG20 exhibits ligand binding properties distinct from the three receptor subtypes identified by molecular cloning. Saturation binding studies indicate an affinity constant of 15 +/- 1.2 nM for the alpha 2-AR antagonist [3H]rauwolscine, a value 6-20 times higher than that observed for the three cloned receptor subtypes. In competition binding studies the potency order of competing ligands for RG20 is phentolamine greater than idazoxan greater than yohimbine greater than rauwolscine greater than prazosin. Of the three previously cloned alpha 2-AR, RG20 is most closely related to the human C-10 alpha 2-AR (89% homology) and is also capable of mediating adenylylcyclase inhibition as determined following its stable expression in NIH-3T3 fibroblasts. However, in contrast to RG20, [3H] rauwolscine exhibits a KD of 2 nM for the C-10 receptor, and the potency order for competing ligands is rauwolscine greater than or equal to yohimbine greater than idazoxan greater than phentolamine greater than prazosin. RG20 and C-10 are also distinguished by their affinity for SKF-10478 (RG20 Ki = 531 nM, C-10 Ki = 101 nM), a compound that may functionally distinguish pre- and postsynaptic alpha 2-AR. These data suggest that RG20 represents a fourth alpha 2-AR subtype distinct from the known alpha 2A-C receptor subtypes.

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. I. Identification by photolabeling or membrane and ternary complex preparation.

The G-protein involved in alpha 1-adrenergic receptor signaling was identified using two different approaches. First, purified rat liver membranes were incubated with [alpha-32P]GTP in the absence or presence of the adrenergic agonist (-)-epinephrine, or in the presence of GTP. After UV irradiation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, covalent labeling of a number of proteins was apparent and could be blocked by unlabeled GTP. In the preparation treated with (-)-epinephrine alone, labeling of a 74-kDa species was markedly enhanced. Enhanced labeling of 40-50-kDa species was also observed. Labeling of the 74-kDa protein was also evident in similarly treated membranes prepared from FRTL-5 thyroid cells, which contain abundant alpha 1-adrenergic receptors, but not in those prepared from turkey erythrocytes or NIH 3T3 fibroblasts, which are essentially devoid of alpha 1-receptors. Second, alpha 1-agonist-receptor-G-protein ternary complex formation was induced by incubating purified rat liver membranes with (-)-epinephrine. Rauwolscine (10(-7) M) and (+/-)-propranolol (10(-6) M) were included to prevent activation of alpha 2- and beta-adrenergic receptors by (-)-epinephrine. The ternary complex of hormone, receptor, and G-protein was solubilized, partially purified using heparin- and wheat germ agglutinin-agarose, and reconstituted into phospholipid vesicles. The vesicles displayed agonist-stimulated guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding that was blocked by phentolamine (10(-4) M). By contrast, stimulation of GTP gamma S binding was not evident when the vesicles were incubated with the beta-agonist, isoproterenol. Incubation of the vesicles with [alpha-32P]GTP or [alpha-32P]azido-GTP in the presence of (-)-epinephrine, followed by photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, resulted in the covalent labeling of a 74-kDa protein. Labeling of this protein could be blocked by preincubation with phentolamine or unlabeled GTP. These findings provide direct evidence for the coupling of the alpha 1-adrenergic receptor to a previously uncharacterized G-protein (termed Gh), which has an apparent molecular mass of approximately 74 kDa.

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution.

In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.

The hydrophobic tryptic core of the porcine alpha 2-adrenergic receptor retains allosteric modulation of binding by Na+, H+, and 5-amino-substituted amiloride analogs.

Extensive trypsinization of the purified alpha 2-adrenergic receptor and repurification by wheat germ agglutinin-agarose chromatography yields an adrenergic ligand-binding hydrophobic core of the receptor. Allosteric modulation of adrenergic ligand binding by Na+, H+, and 5-amino-substituted analogs of amiloride is quantitatively retained in this core, as assessed by the ability of these agents to accelerate the rate of [3H] yohimbine dissociation from the adrenergic ligand-binding site. These findings refine our understanding of where within the alpha 2-adrenergic receptor structure these allosteric agents bind and, for the effects of Na+ and H+, allow certain predictions to be made as to which carboxylic acid side chains are probable candidates for participation in a monovalent cation-binding pocket within the hydrophobic tryptic core of the receptor.

Cloning, sequencing, and expression of the gene encoding the porcine alpha 2-adrenergic receptor. Allosteric modulation by Na+, H+, and amiloride analogs.

The gene for an alpha 2-adrenergic receptor has been cloned from a porcine genomic library, using as a probe a 0.95-kilobase Pst fragment of the gene for the human platelet alpha 2-adrenergic receptor. The identity of the cloned porcine gene was confirmed initially on the basis of partial amino acid sequence information obtained following cyanogen bromide digestion of homogeneous preparations of porcine brain alpha 2-adrenergic receptors. The deduced amino acid sequence for the porcine receptor, when compared to other members of the family of guanine nucleotide-binding protein-coupled receptors, shares the same overall structural characteristics and most closely resembles the human platelet C10 alpha 2-adrenergic receptor (greater than 93% homology). The putative porcine alpha 2-receptor gene was expressed in the COS-M6 cell line. Transfected cells display saturable [3H]yohimbine binding. The KD for [3H]yohimbine, determined in digitonin-solubilized preparations, is 5.8 nM. The selectivity of agonists and antagonists in competing for [3H]yohimbine binding to membranes prepared from the transfected cells is characteristic of the alpha 2A subtype of adrenergic receptors. The porcine alpha 2-receptor also was expressed permanently in LLC-PK1 porcine kidney cells at a level of 100 pmol/mg protein. The alpha 2-agonist UK14304 is able to attenuate forskolin or vasopressin-stimulated cAMP accumulation by at least 50% in these cells. Allosteric modulation of [3H] yohimbine binding by Na+, H+, and 5-amino-substituted analogs of amiloride also was demonstrated for the alpha 2-receptor expressed in COS-M6 cells. Moreover, these modulatory effects were quantitatively similar to those observed for homogeneous preparations of the alpha 2-receptor purified from porcine brain cortex. Retention of the effects of cations and amiloride analogs in transiently expressed alpha 2-receptors supports the interpretation that the allosteric sites for these agents reside in the alpha 2-receptor molecule itself.

Identification and structural characterization of alpha 1-adrenergic receptor subtypes.

Rat liver and brain membrane alpha 1-adrenergic receptors were purified greater than 500-fold by successive chromatographic steps using heparin-agarose, an affinity matrix constructed by coupling a novel derivative of the alpha 1-selective antagonist prazosin to Affigel-102 and wheat germ agglutinin-agarose. Several lines of evidence were obtained for the existence in brain of an alpha 1-adrenergic receptor subtype that is structurally distinct from that previously characterized in liver and other tissues using photoaffinity labeling, protein purification, and DNA cloning techniques. The alpha 1-selective ligand chlorethylclonidine (CEC) (an alkylating agent) irreversibly inactivates 100% of [3H]prazosin binding sites in partially purified preparations of rat liver. Under identical conditions, only 50% of brain receptors are irreversibly inactivated. Computer modeling of data obtained from the competition by the alpha antagonists WB4101 and phentolamine for [3H]prazosin binding to partially purified preparations of rat liver is best fit by assuming a single class of low affinity sites for both ligands. However, analysis of partially purified brain preparations indicates the presence of two binding sites with different affinities for these antagonists. Additionally, prior alkylation of brain receptors with CEC results in the loss of low affinity phentolamine and WB4101 binding sites. The CEC-insensitive site in brain, which displays high affinity for phentolamine and WB4101, is resistant to photoaffinity labeling by [125I]azidoprazosin. This is not due to a markedly lower affinity of the CEC-insensitive sites for the photoaffinity label, because competition studies with [127I]azidoprazosin revealed a single class of high affinity sites in partially purified brain samples. Photoaffinity labeling of partially purified liver and brain samples not treated with CEC results in the specific labeling of a single protein of Mr 80,000. No specifically labeled protein is observed for partially purified brain samples that had previously been incubated with CEC. Treatment of photoaffinity-labeled liver and brain receptors with N-glycanase to cleave N-linked oligosaccharides results in a single Mr 55,000 protein. Taken together, these data provide evidence for the existence of a single receptor subtype (alpha 1b) in rat liver and for two subtypes (alpha 1a and alpha 1b) in rat brain. Furthermore, the insensitivity of the alpha 1a subtype to CEC and the resistance of the alpha 1a subtype to covalent labeling by an alpha 1b-selective photoaffinity probe suggest that the primary structures of the two receptor subtypes differ, such that an amino acid(s) in the alpha 1b subtype that incorporates CEC and the photoaffinity label is lacking in the alpha 1a subtype.

[The identification and characteristics of subpopulations of alpha 1-adrenergic receptors]. / Identifikatsiia i kharakteristika subpopuliatsii al'fa 1-adrenergicheskikh retseptorov.

Rat liver and brain alpha 1-adrenergic receptors were purified 500 fold by successive chromatographic steps using heparin- and wheat germ agglutinin-agarose; an affinity matrix constructed by coupling CP85.224 (a derivative of prazosin) to affigel 102. It is shown that the existence in brain of an alpha 1-adrenergic receptor subpopulation, which is structurally distinct from that previously characterized. Chlorethylclonidine, irreversibly inactivates [3H] prazosin binding sites in partially purified membrane preparations of rat liver. Under identical conditions, only 50% of receptors are irreversibly inactivated. Computer modelling of data obtained from the competition by the alpha-antagonists, WB 4101 and phentolamine, for [3H] prazosin binding to partially purified preparations of rat liver is best fit by assuming a single low-affinity site for both ligands. However, the partially purified brain preparations indicates the presence of two affinity binding sites for these antagonists. Prior alkylation of brain receptors with chlorethylclonydyne results in the loss of the low-affinity phentolamine and WB4101 binding sites. These data provide evidence for the existence of a single receptor subpopulation (alpha 1b) in rat liver and for two subpopulations (alpha 1a and alpha 1b) in rat brain. The significance of these results in understanding the signal mechanisms which allow cellular responsiveness to alpha 1-adrenergic receptor agonists is discussed.

Molecular comparison of alpha 2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelet.

We have previously described a simple two-step purification technique to isolate alpha 2-adrenergic receptors from the rat adrenocortical carcinoma (Jaiswal, R. K. and Sharma, R. K. (1985) Biochem. Biophys. Res. Commun. 130, 58-64). Utilizing this technique we have now achieved approximately 77,000-fold purification to apparent homogeneity of alpha 2-adrenergic receptors from human platelets. We have compared the biochemical characteristics of these receptors with those from the rat, which were purified approximately 40,000-fold to homogeneity. The [125I] receptor proteins from two sources showed: (a) a single radioactive band with a Mr of 64,000 as evidenced by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); and (b) a single symmetrical peak with a pI of 4.2 by isoelectric focusing polyacrylamide gel electrophoresis. Both proteins showed typical alpha 2-adrenergic binding characteristics with specific binding activities of 13.85 nmol/mg and 14.17 nmol/mg protein. These values are close to the theoretical binding activity of 15.6 nmol/mg protein for 1 mol of the ligand binding 1 mol of the receptor protein. These results attest to the purity of the receptors, to its Mr of 64,000, and to its acidic nature. However, the peptide maps of the radioiodinated alpha 2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelets reveal some distinct differences which may relate to the differences in the pharmacological specificities between rodent and non-rodent alpha 2-adrenergic receptors.

Glycerol, sodium phosphate, and sodium chloride permit the solubilization and partial purification of rat hepatic alpha 1-receptors by 3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate.

CHAPS [3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate], a zwitterionic detergent, has been used to solubilize the rat hepatic alpha 1-adrenergic receptor. Although the use of this detergent alone permitted a poor receptor solubilization, the inclusion of sodium phosphate, sodium chloride, and glycerol to the medium allowed 30% of the binding activity observed in plasma membranes to be recovered. Binding of the selective alpha 1-adrenergic antagonist, [3H]prazosin, by the solubilized preparation was saturable and of high affinity. In addition, binding of the radioligand was inhibited by a variety of adrenergic agents with affinity, specificity, and stereoselectivity comparable to that observed in plasma membranes. The use of glycerol in the solubilization medium permitted recovery of the solubilized receptor in a stable form (T1/2 = 72 h at 4 degrees C). Sequential affinity and size-exclusion gel chromatography allowed a 1000-fold purification of the solubilized receptor. The Stokes' radius and the apparent molecular mass of the purified receptor-Chaps complex (48.4 A and 160,000 Da, respectively), determined by gel filtration chromatography, were similar to those previously obtained for the rat hepatic alpha 1-receptor purified after solubilization with the nonionic detergent digitonin. These data indicate that the combination of Chaps, sodium phosphate, sodium chloride, and glycerol permitted the solubilization and partial purification of hepatic alpha 1-receptor in an active and stable form. The use of this technique might be useful for the solubilization of other membrane-bound proteins by Chaps whose biophysical characteristics make it an ideal detergent for reconstitution experiments.

Characterization of alpha 1-adrenergic receptors in perfused rat heart.

The characteristics of alpha 1-adrenergic receptors were investigated in perfused rat hearts at 37 degrees C. [3H]Prazosin was bound in a time-dependent manner and reached equilibrium at 15 min. Scatchard analysis of the specific binding isotherm for [3H]prazosin indicated a population of high affinity sites (Kd = 0.41 nM, Bmax = 13.2 pmol/g wet wt). Prazosin binding was displaced by epinephrine as well as by the adrenergic antagonists prazosin greater than phentolamine greater than yohimbine greater than propranolol. Specific prazosin binding was defined as that portion of the binding inhibited by 10 microM phentolamine; phentolamine and epinephrine displaced 3H-prazosin to the same level. [3H]Prazosin was not metabolized by the heart. When pre-labelled hearts were perfused at 37 degrees C with prazosin-free medium non-specific binding of [3H]prazosin decreased more rapidly (t0.5 = 4 min) than specific binding (t0.5 = 38 min). Perfusion of the heart at lower temperatures (less than 10 degrees C) decreased the rate of loss of nonspecific binding and prevented the loss of specific binding. Fractionation of [3H]prazosin perfused hearts at 0 degrees C, when dissociation was minimal, led to a loss of binding so that sarcolemma-enriched fractions contained approximately 2% of the binding sites present in the perfused heart. The binding characteristics of sarcolemma-enriched fractions (Kd 0.10 nM, Bmax 300 fmol/mg protein) differed significantly from those of the perfused heart. Exposure of the heart to 10 min of ischaemia prior to binding studies did not alter the characteristics of the [3H]prazosin binding sites. It is concluded that the perfused rat heart contains a population of alpha 1-adrenoceptors which differ from those of isolated sarcolemma preparations perhaps because of alterations that occur during sarcolemma isolation. The perfused heart should be an appropriate model system in which to study the relationship between receptor occupancy and biological response as well as the direct effects of perturbations such as ischaemia.

Glycoprotein nature of alpha 2-adrenergic receptors labeled with p-azido[3H] clonidine in calf retina membranes.

alpha 2-Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated agonist p-azido[3H]clonidine. Saturation binding in the dark occurs with high affinity (1.3 +/- 0.3 nM) to a single class of sites (1122 +/- 67 fmol/mg protein). Irradiation of the membrane-bound radioligand results in the labeling of a peptide band with an apparent size of 65 kDa and a characteristic pharmacological profile for an alpha 2-adrenergic receptor. The carbohydrate moieties of the alpha 2-receptor are characterized by lectin affinity chromatography and glycosidase treatment. The Nonidet P-40-solubilized, p-azido[3H]clonidine-labeled receptors are completely retained by Con A- as well as WGA-Sepharose columns. Neuraminidase, alpha-mannosidase and TFMS do not affect the electrophoretic mobility of the receptor on SDS-PAGE whereas endoglycosidase F reduces the apparent size to 45 kDa.

Separation of solubilized A2 adenosine receptors of human platelets from non-receptor [3H]NECA binding sites by gel filtration.

Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate) and the solubilized extract subjected to gel filtration. Binding of the adenosine receptor agonist [3H]NECA (5'-N-ethylcarboxamidoadenosine) was measured to the eluted fractions. Two [3H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the [3H]NECA binding activity eluted from the column. It bound [3H]NECA in a reversible, saturable and GTP-dependent manner with an affinity of 46 nmol/l and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of [3H]NECA to the first peak with a pharmacological profile characteristic for the A2 adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for [3H]NECA binding to the second, major peak. These results suggest that a solubilized A2 receptor-Gs protein complex of human platelets can be separated from other [3H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A2 adenosine receptor of human platelets.

Glycosylation of the mammalian alpha 1-adrenergic receptor by complex type N-linked oligosaccharides.

The binding subunit of the alpha 1-adrenergic receptor has been identified as an Mr = 80,000 peptide in several tissues. Adsorption of the alpha 1-adrenergic receptor to a wheat germ agglutinin lectin-agarose resin suggests that the receptor protein is glycosylated. In this study, we investigated the nature of the carbohydrate chains linked to the alpha 1-adrenergic receptor peptide. The alpha 1-adrenergic receptor from DDT2 MF-2 smooth muscle cell and rat brain membranes was photolabeled with 125I-azido-prazosin [( 125I]CP65,526) and then treated with exoglycohydrolases prior to SDS-PAGE and autoradiography. Removal of terminal sialic acid residues by neuraminidase decreased the receptor Mr by 6,000; however, alpha-mannosidase was without effect, indicating complex type glycosylation of the receptor-protein. Similar results were observed for the rat hepatic membrane alpha 1-adrenergic receptor. Removal of N-linked carbohydrates at asparagine residues by peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase (from Flavobacterium meningosepticum) resulted in a specifically labeled peptide at Mr = 50,000-55,000 in DDT1 MF-2 membrane and solubilized receptor preparations. Treatment of DDT1 MF-2 cells with swainsonine or (+)-1-deoxymannojirimycin, inhibitors of complex type carbohydrate chain biosynthesis, caused a reduction in the apparent molecular weight of the receptor (Mr = 60,000) but did not alter the number of alpha 1-adrenergic receptors per cell or their affinity for the radioligand [3H]prazosin. These findings indicate that the alpha 1-adrenergic receptor is heavily glycosylated, the major oligosaccharide moiety being of the complex type, N-linked to asparagine residues. The peptide backbone of the receptor has an Mr less than or equal to 55,000, consistent with the predicted molecular mass of other membrane neurotransmitter receptors based on sequence analysis of isolated cDNA clones.

Purification of the alpha 2-adrenergic receptor from porcine brain using a yohimbine-agarose affinity matrix.

A procedure has been developed for purification of the porcine brain alpha 2-adrenergic receptor to homogeneity. alpha 2-Adrenergic receptors were solubilized from porcine brain particulate preparations using sequential extraction into sodium cholate- and digitonin-containing buffers. The alpha 2-adrenergic receptors in the digitonin extract were identified using the alpha 2-adrenergic selective antagonist, [3H]yohimbine, and demonstrated the same specificity for interaction with adrenergic ligands as did the receptors in particulate preparations. Extraction into digitonin-containing buffers eliminated the modulation of receptor-agonist interactions by guanine nucleotides, but not by monovalent cations. A novel affinity resin, yohimbine-agarose, was synthesized and used for purification of alpha 2-adrenergic receptors. Using two sequential yohimbine-agarose affinity chromatography steps, digitonin-solubilized alpha 2-adrenergic receptors from porcine brain cortex were purified to homogeneity as assessed by radioiodination and silver stain analysis of these preparations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified alpha 2-adrenergic receptor has an approximate Mr = 65,000, as determined by photolabeling of the adrenergic ligand-binding subunit. The yohimbine-agarose affinity resin should be useful for purifying quantities of receptor sufficient for studies of receptor structure and function.

Membrane reconstitution of high-affinity alpha 2 adrenergic agonist binding with guanine nucleotide regulatory proteins.

Regulation of adenylate cyclase by alpha 2 adrenergic receptors requires the inhibitory guanine nucleotide binding protein Ni. A role for this protein has also been suggested in the high-affinity binding of agonists to the alpha 2 receptor. We recently reported that alkaline treatment can selectively inactivate alpha 2 agonist binding and Ni in human platelet plasma membranes [Kim, M.H. & Neubig, R.R. (1985) FEBS Lett. 192, 321-325]. Binding of the full alpha 2 agonists epinephrine and 5-bromo-6-[N-(4,5-dihydroimidazol-2-yl)amino]quinoxaline (UK 14,304) to these membranes was determined by competition and direct radioligand binding, respectively. The high-affinity GTP-sensitive binding of the agonists is lost after alkaline treatment. Binding of [3H]UK 14,304 was reconstituted by poly(ethylene glycol)-induced fusion of alkaline-treated platelet membranes with cell membranes containing Ni but no alpha 2 receptor or with lipid vesicles containing purified guanine nucleotide binding proteins (N-proteins) from bovine brain. The reconstituted binding was of high affinity (Kd = 0.4 +/- 0.1 nM), accounted for a substantial fraction of the total alpha 2 receptors (Bmax for [3H]UK 14,304 was 78 +/- 23% of the Bmax for [3H]yohimbine), and was abolished in the presence of guanosine 5'-(beta, gamma-imidotriphosphate) (GppNHp). The brain-specific protein No (predominant guanine nucleotide regulatory protein from bovine brain) was also effective in reconstituting high-affinity alpha 2 agonist binding. The results presented here show that a guanine nucleotide regulatory protein of the No or Ni type is necessary for high-affinity alpha 2 agonist binding. These methods should also prove useful for future studies of receptor N-protein interactions.