Alpha adrenergic receptors in the rabbit bladder base smooth muscle: alpha-1 adrenergic receptors mediate contractile responses

Alpha adrenergic receptors in rabbit bladder base smooth muscle were investigated by in vitro responses of smooth muscle strips to exogenous alpha agonist stimulation and in radioligand binding assays. Norepinephrine and phenylephrine caused significantly greater maximal contractile responses than did clonidine. Also, the contractile response was only inhibited weakly by the alpha-2 selective antagonist yohimbine but was potently inhibited by alpha-1 selective antagonists prazosin and BE2254, suggesting that the response is mediated predominantly by alpha-1 adrenergic receptors. The alpha-1 selective antagonist [125I]BE2254 was used to specifically label a single class of binding sites with a dissociation constant of 131.0 +/- 5.9 pM and a maximal binding capacity of 17.6 +/- 1.9 fmol/mg of protein. Catecholamines compete for [125I]BE2254 binding stereospecifically and with the characteristic alpha adrenergic potency series of (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The alpha-1 selective antagonist prazosin (Kd = 2.4 nM) is much more potent in competing for [125I]BE2254 binding than is the alpha-2 selective antagonist yohimbine (Kd = 2900 nM). Also, this dissociation constant of prazosin of [125I]BE2254 binding for bladder base smooth muscle membranes was similar to prazosin's pA2 value of 8.23 to 8.58 in the contraction experiments. The results suggest that alpha-1 rather than alpha-2 receptors predominantly mediate catecholamine-induced contraction in the rabbit bladder base. Also, these receptor sites can be measured directly with the specific antagonist radioligand, [125I]BE2254.     

Classification of bovine aortic alpha-1 adrenoceptors by the criteria of ligand affinity and molecular mass

[3H]Prazosin bound to a single class of alpha-1 adrenoceptors in bovine aortic membranes with a Kd of 25 pM. Digitonin solubilized 30% of the receptors as assayed by specific [3H] prazosin binding. The rank order potency of displacing ligands was the same for both membrane-bound and soluble alpha-1 adrenoceptors [prazosin greater than phentolamine greater than yohimbine and (-)-epinephrine = (-)-norepinephrine much greater than (+)-norepinephrine]. Prazosin had a significantly lower affinity for the soluble receptor, whereas the other adrenergic ligands had the same affinity for both forms of the receptor. The alpha-1 adrenoceptor was affinity-labeled with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7- dimethoxyquinazoline and analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the receptor binding subunit in both bovine aorta and rat liver was found to be 86,000 MW. It is concluded from this study that bovine aortic alpha-1 adrenoceptors have pharmacologic and biochemical characteristics similar to those in other tissues.     

Characterization of alpha-1 adrenergic receptors in the heart using [3H]WB4101: effect of 6-hydroxydopamine treatment

To identify and characterize the cardiac alpha-adrenoceptors, a radioreceptor binding assay using the potent alpha adrenergic antagonist, [3H]WB4101 was performed in rat hearts. Specific [3H]WB4101 binding to rat left ventricular homogenates was saturable, reversible and of high affinity (Kd = 0.18 nM) with a Bmax of 2.57 fmol/mg of tissue (27.7 fmol/mg of protein). Adrenergic agonists competed for specific [3H]WB4101 binding in the order: (-)-epinephrine > (-)-norepinephrine greater than (-)-isoproterenol. Stereospecificity of the [3H]WB4101 binding sites was also demonstrated with (-)-epinephrine, (Ki = 90) nM being 270 times as potent as (+)-epinephrine, (K1 = 24 microM). Adrenergic antagonists competed for the binding in the order: WB4101 = prazosin greater than yohimbine greater than (-)-propranolol. WB4101 and prazosin exhibited a markedly greater (2000 times) affinity for [3H]WB4101 binding sites than yohimbine. The affinities (pKi) of alpha agonists and antagonists for [3H]WB4101 binding sites in the rat heart closely correlated with their pharmacological potencies in the heart. Scatchard analysis for [3H]WB4101 binding, performed in five regions from control and 6-hydroxydopamine-treated rat hearts, revealed specific [3H]WB4101 binding (Bmax) significantly greater in the ventricles and intraventricular septae than in atria. At 1 week after 6-hydroxydopamine treatment, there was a significant increase (40%) in the Bmax for [3H]WB4101 binding to ventricles and intraventricular septae without a change in Kd. We conclude: 1) [3H]Wb4101 selectively labels postsynaptic alpha-1 adrenoceptors in the rat heart; 2) there is a definite regional variation for cardiac alpha-1 adrenoceptors; and 3) 6-hydroxydopamine treatment caused a significant increase in the density of alpha-1 adrenoceptors in ventricles and intraventricular septae, compatible with a postsynaptic localization of the [3H]WB4101 binding site.     

Ability of SK&F 104078 and SK&F 104856 to identify alpha-2 adrenoceptor subtypes in NCB20 cells and guinea pig lung.

Alpha-2 adrenoceptors were characterized in three tissue culture cell lines and in membrane homogenates of guinea pig lung via the ability of a series of alpha adrenoceptor antagonists to inhibit the binding of [3H]clonidine, [3H]UK-14,304 and [3H]rauwolscine. The cells studied included those known to possess receptors of alpha-2A (HT29) and alpha-2B (NG108-15) subtypes as well as the previously uncharacterized NCB20 cells. Correlation of the ability of the antagonists to inhibit [3H]clonidine or [3H]UK-14,304 binding did not identify alpha-2 adrenoceptor subtypes. On the other hand, correlation of antagonist affinities against [3H]rauwolscine binding showed HT29 cells and guinea pig lung to have similar characteristics (r = 0.911) as did NG108-15 and NCB20 cells (r = 0.985). These data suggest subtle differences in the binding of [3H]agonists and [3H]antagonists to the alpha-2 adrenoceptor, resulting in the failure of [3H]clonidine and [3H]UK-14,304 to recognize differences between alpha-2A and alpha-2B receptor subtypes. 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SK&F 104078) did not differentiate between the alpha-2A and alpha-2B receptor subtypes. However, 2-vinyl-7-chloro-3,4,5,6-tetrahydro-4-methylthieno[(4,3,2ef] [3]benzazepine (SK&F 104856), which has similar selectivity in functional in vitro models, was about 35-fold more potent in displacing [3H]rauwolscine binding to the alpha-2B site. These data provide additional evidence that the functional subclassification of alpha-2 adrenoceptors based on sensitivity to SK&F 104078 and SK&F 104856 subdivides this receptor in a different manner than does the alpha-2A/alpha-2B subclassification scheme.     

Napamezole, an alpha-2 adrenergic receptor antagonist and monoamine uptake inhibitor in vitro

Napamezole (2-[3,4-dihydro-2-naphthalenyl)methyl]-4,5-dihydro-1H- imidazole-monohydrochloride) is a selective alpha-2 adrenergic receptor antagonist and a monoamine re-uptake inhibitor in vitro. The alpha adrenergic antagonist activity of napamezole was determined in vitro in rat brain receptor binding assay using [3H]clonidine and [3H]prazosin for alpha-2 and alpha-1 receptors, respectively. The Ki values for napamezole were 28 nM (alpha-2) and 93 nM (alpha-1). The relative potencies for inhibiting [3H]clonidine binding were: phentolamine greater than idazoxan greater than napamezole greater than mianserin greater than yohimbine greater than piperoxan greater than rauwolscine greater than tolazoline much greater than prazosin; and for inhibition [3H]prazosin binding they were: prazosin greater than phentolamine greater than mianserin greater than napamezole greater than yohimbine greater than idazoxan greater than tolazoline. Alpha adrenoceptor antagonism was also assessed in the isolated rat vas deferens. Napamezole reversed clonidine-induced decreased in twitch height in the electrically stimulated rat vas deferens (alpha-2 antagonism with a Kb of 17 nM). The rank order of potency as an alpha-2 antagonist relative to other compounds was phentolamine greater than idazoxan greater than yohimbine greater than piperoxan = napamezole greater than mianserin much greater than prazosin. Napamezole also antagonized methoxamine-induced contractions (alpha-1) of the rat vas deferens with a Kb of 135 nM. The rank order of potency of these compounds as alpha-1 antagonists was prazosin greater than phentolamine greater than mianserin greater than yohimbine greater than napamezole greater than idazoxan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anatomical evidence for alpha-2 adrenoceptor heterogeneity: differential autoradiographic distributions of [3H]rauwolscine and [3H]idazoxan in rat brain

Previous autoradiographic studies which have localized alpha-2 adrenoceptors within brain have used tritiated derivatives of clonidine and other alpha-2 adrenergic agonists. In the present study we have compared the autoradiographic distributions of binding sites labeled by two reportedly selective alpha-2 adrenoceptor antagonists, [3H]rauwolscine and [3H]idazoxan, in rat brain. The distribution of high affinity [3H]idazoxan binding sites differed markedly from that of high affinity [3H]rauwolscine sites throughout the neuraxis. The distribution of [3H]idazoxan binding sites paralleled closely that of [3H]clonidine sites, and corresponded to areas of noradrenergic innervation. Densest [3H]idazoxan labeling appeared over anterior olfactory nuclei, fundus striatum, septum, thalamus, hypothalamus, amygdala, entorhinal cortex, central gray, inferior colliculus, dorsal parabrachial nucleus, locus ceruleus and nucleus of the solitary tract. In contrast, much lower levels of [3H]rauwolscine labeling appeared over several areas which receive primarily dopaminergic innervation, and thus corresponded closely to [3H]spiroperidol binding distributions. Densest [3H]rauwolscine labeling appeared over nucleus caudate-putamen, nucleus accumbens, olfactory tubercle, Islands of Calleja, hippocampus, parasubiculum, basolateral amygdaloid nucleus and substantia nigra. In areas labeled by [3H]rauwolscine, computerized densitometric analysis of autoradiographic saturation curves revealed that the maximum binding values for [3H]idazoxan high affinity binding sites were consistently greater than those for high affinity [3H]rauwolscine sites. The pharmacological characterization of these two binding sites in the accompanying paper supports the present anatomical evidence that [3H]idazoxan labels a heterogenous population of alpha-2 adrenoceptor sites, one population of which is selectively labeled by [3H]rauwolscine.     

Human fat cell alpha-2 adrenoceptors. II. Comparative study of partial and full agonist binding parameters using [3H]clonidine and [3H]UK-14,304

Binding studies were carried out on human fat cell membranes with the major radioligands available for alpha-2 adrenergic receptor identification: the antagonist [3H]yohimbine, the partial agonist [3H]clonidine ([3H]CLO) and the full agonist radioligand [3H]UK-14,304 ([3H]UK). Binding approaches performed with [3H]UK and [3H]CLO; two imidazoline derivatives exhibiting full and partial agonist properties, respectively, in biological assays clearly indicate that: 1) partial and full agonists label an equivalent number of binding sites corresponding to the high affinity form of the alpha-2 receptor; 2) there is some correlation between the KiH/KiL ratio defined in competition of [3H]yohimbine binding and the intrinsic activity defined in biological assays; 3) differences exist between the dissociation of the full-agonist ([3H]UK) and the partial-agonist ([3H]CLO); 4) the interaction of the full agonist with the alpha-2 receptor promotes the formation of an agonist-alpha-2 receptor-Gi protein complex (HRGi) complex which is more stable than that obtained with the partial agonist as objectivated by the sensitivity to the effects of guanosine 5'-(imido)triphosphate and N-ethylmaleimide; 5) the full-agonist is characterized by a "tight agonist binding" which is not observed with the partial agonist. From a functional point of view, the lower biological activity of the partial alpha-2 agonist could be explained by the formation of more labile HRGi complexes having weaker stability by comparison with the full agonist agents which promote stronger HRGi complexes and sustained activity.     

Pharmacological characterization of alpha adrenergic receptors in the young and old female rabbit urethra.

The pharmacological characteristics of alpha-1 and alpha-2 adrenergic receptors in young (6 month) and old (4.5-5 year) female rabbit urethra were studied using isolated muscle bath techniques. Norepinephrine, phenylephrine, clonidine, oxymetazoline and UK 14,304 produced concentration-dependent contractions in both age groups. The maximum contractile responses (Emax) to norepinephrine, phenylephrine, oxymetazoline and UK 14,304 were of similar magnitude and were significantly greater than the contractile responses to clonidine. The rank order of the ED50 values for these drugs was: oxymetazoline less than UK 14,304 much less than clonidine = norepinephrine = phenylephrine. Prazosin (10(-8) M) shifted the concentration-response curves to phenylephrine and UK 14,304 to the right, but did not shift the concentration-response curves to clonidine and oxymetazoline. Yohimbine (10(-7) M) shifted the concentration-response curves to clonidine, oxymetazoline and UK 14,304 to the right, but did not shift the concentration-response curve to phenylephrine. The ED50 values for phenylephrine and clonidine were smaller in the older than in the younger age group. There were no other age-dependent differences in the response to agonists. Pretreatment with chlorethylclonidine, which selectively alkylates the alpha-1B subtype, did not affect the Emax value of phenylephrine-induced contractions, but significantly shifted the curve to the right. The ratios of the Emax values in Ca++ free buffer to that in normal Ca++ buffer for phenylephrine, UK 14,304, clonidine, oxymetazoline and KCl were 0.30, 0.38, 0.08, 0.07 and 0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-2 adrenoceptor in rat jejunum epithelial cells: characterization with [3H]RX821002 and distribution along the villus-crypt axis

Alpha-2 adrenergic receptivity of rat jejunum epithelial cells was studied using the new antagonist radioligand, [3H]RX821002 [( 3H]-2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline). All the parameters of [3H]RX821002 binding were consistent with the labeling of an alpha-2 adrenoceptor. The use of this probe was moreover extremely convenient, because contrarily to [3H]yohimbine and [3H]rauwolscine, [3H]RX821002 displayed in this tissue a very high affinity (Kd = 0.54 +/- 0.12 nM) and a low level of nonspecific binding (5% at 1 nM [3H]RX821002). Competition studies with various antagonists and agonists showed that the labeled sites were alpha-2-selective and stereospecific. Oxymetazoline was much more potent than chlorpromazine or prazosin suggesting that the receptor is of the alpha-2-subtype. Yohimbine and rauwolscine were equipotent, which is also in agreement with the pharmacological definition of this subtype. These two compounds displayed, however, a rather weak affinity (Ki approximately 40 nM), which is somewhat different with what one should expect for a true alpha-2A adrenoceptor. Altogether the competition data indicated that the alpha-2 adrenoceptor from rat jejunal epithelium is neither an alpha-2A, nor an alpha-2B, nor an alpha-2c adrenoceptor and may belong to a fourth subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

New tools for human fat cell alpha-2A adrenoceptor characterization. Identification on membranes and on intact cells using the new antagonist [3H]RX821002

The pharmacology of the alpha-2 adrenoceptor of the human adipocyte was improved by using some new alpha-2 antagonists from different chemical families (imidazolines, benzazepines and benzofuroquinolizines) in biological and binding assays. Moreover, investigations were also carried out to define the binding properties of a new imidazolinic antagonist, RX821002 [2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline], which could be a potential radioligand. [3H]RX821002 binding was very rapid and reversible. Saturation isotherms indicated that [3H]RX821002 labeled, with high affinity, a homogeneous population of noninteracting binding sites with a mean Kd of 0.98 +/- 0.05 nM (n = 6). The binding of [3H]RX821002 on the human fat cell alpha-2 adrenoceptor displayed a specificity which is strictly similar to that obtained with [3H]rauwolscine and which is classical for an alpha-2 A adrenoceptor. The binding parameters of [3H]RX821002 were compared with those obtained with the classical alpha-2 antagonist [3H]yohimbine. Analysis of the data indicate: 1) that [3H]RX821002 exhibited higher affinity; 2) that the nonspecific binding of [3H]RX821002 was very low; 3) that the total number of sites (maximum binding values) defined with [3H]RX821002 was significantly higher than that defined with [3H]yohimbine. This difference was not due to a specific preferential labeling of one of the two affinity states of the receptor, but suggested that [3H]yohimbine does not label the whole receptor population; 4) that [3H]RX821002 specific binding was less sensitive to magnesium chloride and GTP than [3H]yohimbine binding; and 5) that [3H]RX821002 can be used suitably for identification of alpha-2 adrenoceptors on the intact adipocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of adrenoceptor subtypes in cat cutaneous vasculature

Experiments were undertaken to characterize the relative contribution of adrenoceptor subtypes in mediating vasoconstriction to exogenous agonists in the digital cutaneous vascular bed of the anesthetized cat using laser-Doppler flowmetry. Intra-arterial administration of (-)-epinephrine and (-)-norepinephrine into the brachial artery caused a dose-related vasoconstriction (decreased flow) with ED50 values of 7 and 21 ng, respectively. Blockade of beta adrenoceptors with propranolol did not alter the response to (-)-epinephrine nor did i.a. isoproterenol produce a significant vasodilation. Vasoconstrictor responses elicited by (-)-epinephrine and (-)-norepinephrine were antagonized by treatment with phentolamine (2.5 mg/kg i.v.) and by yohimbine (0.5 mg/kg i.v.) but were only marginally blocked by prazosin (0.1 mg/kg i.v.). A dose-related depression of cutaneous blood flow was also caused by clonidine at doses virtually identical to those of (-)-norepinephrine. Clonidine-induced vasoconstriction was antagonized by rauwolscine (0.5 mg/kg i.v.) but not by prazosin (0.1 mg/kg i.v.). Dose-response curves to a variety of additional adrenoceptor stimulants were constructed with the potency rank order for all agonists being: (-)-epinephrine greater than B-HT 920 = (-)-norepinephrine = clonidine much greater than (-)-phenylephrine much greater than B-HT 933 greater than methoxamine. Treatment with prazosin (0.1 mg/kg i.v.) antagonized methoxamine induced cutaneous vasoconstriction but not the decreased blood flow caused by B-HT 933. In contrast, rauwolscine (0.5 mg/kg i.v.) blocked the responses to B-HT 933 but not methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological evidence for alpha-2 adrenoceptor heterogeneity: differential binding properties of [3H]rauwolscine and [3H]idazoxan in rat brain

In the preceding paper, we have reported that the two alpha-2 adrenoceptor antagonists, [3H]rauwolscine and [3H]idazoxan, exhibit markedly different autoradiographic distributions throughout rat brain. Although [3H]idazoxan labeling appears over brain regions receiving noradrenergic innervation, [3H]rauwolscine binding sites are localized most densely in several areas corresponding to dopaminergic terminal fields. We have presently characterized the pharmacological binding properties of high affinity [3H]rauwolscine and [3H]idazoxan labeled sites, using tissue preparation and incubation protocols which are identical to those used in the previous autoradiographic study. Endogenous monoamines inhibited radioligand binding with a rank order of potency of epinephrine = norepinephrine greater than dopamine greater than serotonin. Numerous dopaminergic compounds failed to inhibit either [3H]rauwolscine or [3H]idazoxan binding with high potency, and rauwolscine was a poor inhibitor of [3H]spiroperidol binding. Several adrenergic compounds which selectively label alpha-1 or beta adrenoceptors also exhibited low potency in inhibiting either radioligand. In contrast, alpha-2 adrenoceptor agonists and antagonists possessed high affinity for both [3H]rauwolscine and [3H]idazoxan labeled sites. Their relative potencies at the two sites differed, however. Whereas idazoxan was equipotent in inhibiting either [3H]rauwolscine or [3H]idazoxan binding, rauwolscine exhibited 10-fold higher affinity for its own labeled site. These pharmacological data are consistent with anatomical data presented in the preceding paper, which support the existence of a heterogenous population of alpha-2 adrenoceptors within rat brain, labeled entirely by [3H]idazoxan and only in part by [3H]rauwolscine.     

The insulin-secreting cell line, RINm5F, expresses an alpha-2D adrenoceptor and nonadrenergic idazoxan-binding sites.

The pharmacological properties of alpha-2 adrenoceptors and the existence of nonadrenergic idazoxan-binding sites (NAIBS) were investigated in the insulin-secreting cell-line, RINm5F, using [3H]RX821002 and [3H]idazoxan. Analysis of [3H]RX821002 saturation isotherms revealed the presence of a single class of binding sites (Bmax = 47.5 +/- 3.5 fmol/mg protein) having high affinity (Kd = 1.26 +/- 0.18 nM). Inhibition of [3H]RX821002 binding by adrenergic compounds showed that the labeled sites displayed the properties expected for an alpha-2 adrenoceptor. Based on competition data with drugs having alpha-2 adrenoceptor subtype selectivity, the receptor from RINm5F is neither an alpha-2B nor an alpha-2C. It resembles the alpha-2A, but deviates from this subtype because of a weak affinity for yohimbine and rauwolscine. In this respect, RINm5F alpha-2 adrenoceptor is identical to the receptor previously described in rat intestinal mucosa and corresponds to a fourth subtype: alpha-2D. Agonist inhibition curves were better fitted by a two-site model and indicated that about half of the receptor population was under a high-affinity state corresponding to G protein-coupled receptors. [32P]ADP-ribosylation with pertussis toxin and immunodetection with specific antibodies permitted the identification of three distinct G proteins: Gi2, Gi3 and G0. Binding experiments with [3H]idazoxan showed that this imidazoline labeled two types of sites corresponding to alpha-2 adrenoceptors and NAIBS. Analysis of saturation isotherms under binding conditions allowing to discriminate between the two site populations indicated that the density of NAIBS (44 +/- 2 fmol/mg protein) was fairly identical to that of alpha-2 adrenoceptors. The pharmacological properties of NAIBS, as assessed by determining the relative affinity of imidazolinic and nonimidazolinic compounds, reasonably matched that reported in other tissues. Taken together, these data make the RINm5F cell-line 1) the first model in permanent culture known as expressing an alpha-2 adrenoceptor of the alpha-2D subtype; 2) a good system for studying in vitro the respective role of alpha-2 adrenoceptors and NAIBS in the regulation of insulin secretion by beta cells.     

Pharmacological characterization of alpha adrenoceptors in the rat gastric fundus

Alpha receptors on the intramural cholinergic neurons and on the smooth muscle cells are involved in the inhibitory effect of catecholamines on rat gastric fundus motility. The pharmacological characteristics of these alpha receptors were assessed using longitudinal muscle strips of the rat gastric fundus, contracted to a similar degree by electrical stimulation and by methacholine. All alpha agonists studied (norepinephrine, phenylephrine, methoxamine, clonidine, UK-14,304 and B-HT 920) concentration-dependently inhibited the stimulation-induced contractions. Norepinephrine, phenylephrine, methoxamine and clonidine also inhibited the methacholine-induced contractions, but for the same concentration of agonist, the inhibition was less pronounced than during electrical stimulation-induced contractions; UK-14,304 and B-HT 920 inhibited the methacholine-induced contractions only in a concentration of 10(-4) M. The effect of clonidine and UK-14,304 on electrical stimulation-induced contractions was antagonized competitively by the alpha antagonists rauwolscine and yohimbine (slope in the Schild plot not different from 1). The effect of norepinephrine and phenylephrine on methacholine-induced contractions was antagonized by the alpha antagonists prazosin, corynanthine and yohimbine; against phenylephrine, the antagonism was competitive (slope in the Schild plot not different from 1). It is concluded that the muscular alpha receptors in the rat gastric fundus are of the alpha-1-type. On the postganglionic cholinergic neurons, alpha-2-like receptors are present; it is not yet clear whether the pronounced effect of alpha-1 agonists on the cholinergic neuron activity is due to interaction with these receptors.     

Ablation of the myenteric plexus impairs alpha but not beta adrenergic receptor-mediated mechanical responses of rat jejunal longitudinal muscle

The mechanical responses produced by alpha and beta adrenergic receptor agonists were evaluated in control and myenteric neuron-ablated rat jejunal longitudinal muscle. The myenteric plexus of the jejunum was destroyed by serosal application of benzalkonium chloride (BAC). The beta adrenergic receptor agonists isoproterenol and sulfonterol produced a concentration-dependent relaxation of both control and BAC-treated jejunum. Dose-response curves obtained in control and BAC-treated jejunum were nearly superimposable regardless of the beta agonist used. Isoproterenol-induced relaxation was antagonized by the beta receptor antagonists propranolol and practolol but not by butoxamine. The alpha-1 selective agonists phenylephrine and methoxamine were more potent and efficacious in producing relaxation of control than BAC-treated jejunum. The relaxant responses of methoxamine and phenylephrine in control jejunum were blocked by prazosin but not by yohimbine. The supposed alpha-2 selective agonist clonidine also produced a concentration-dependent, prazosin-sensitive, yohimbine-resistant relaxation which was markedly greater in control than BAC-treated jejunum, consistent with alpha-1 receptor stimulation. Clonidine tested in the presence of prazosin and the alpha-2 selective receptor agonists UK-14,304, M-7 and B-HT 920 all produced a concentration-dependent contraction of control but not BAC-treated jejunum. The contractile response produced by UK-14,304 was antagonized by yohimbine but not by atropine. Our results suggest that in rat jejunal longitudinal muscle: beta adrenergic receptors mediate relaxation and are located on the smooth muscle; alpha-1 adrenergic receptors mediate relaxation and are located on both the smooth muscle and myenteric plexus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-1 adrenergic receptor binding in aortas from rat and dog: comparison of [3H]prazosin and beta-iodo-[125I]-4-hydroxyphenyl-ethyl-aminomethyl-tetralone

We have identified and characterized pharmacologically alpha-1 adrenergic receptors in aorta from dog and rat utilizing two alpha-1 antagonists, [3H]prazosin and beta-3-iodo[125I]-4-hydroxyphenyl-ethyl-aminoethyl-tetralone [125I]HEAT, to determine receptor number and affinity. The rat and dog alpha-1 receptors were found to be very similar. In the dog, KD values were 27 and 11 pM for [3H]prazosin and [125I]HEAT, respectively, whereas maximum binding values were 89 and 65 fmol/mg of protein. KD values in rat aorta were 22 and 15 pM for [3H]prazosin and [125I]HEAT, respectively, whereas maximum binding values were 62 and 47 fmol/mg of protein. Both tissues demonstrated a rank order potency of antagonist inhibition of the two radioligands consistent with an alpha-1 adrenergic receptor with prazosin greater than phentolamine greater than yohimbine. A comparison between IC50 values for both the ligands and the tissues shows strong correlations suggesting the two radioligands are identifying a similar receptor population and that the receptor labeled by the ligands has similar pharmacologic characteristics in the two tissues.     

Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of [3H]prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate [Gpp(NH)p]. A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. [3H]Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with [3H]prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of desensitization of alpha-2-mediated inhibition of lipolysis in fat cells after acute and chronic treatment with clonidine

The regulation of alpha-2 adrenoceptors and alpha-2 adrenergic responses by alpha-2 agonist treatment was investigated in in vitro and in vivo conditions. The alpha-2 adrenergic responsiveness of the adipocytes was tested with an alpha-2 agonist, clonidine, which inhibits, in a concentration-dependent manner, the lipolytic activity of the isolated fat cells incubated in the presence of adenosine deaminase (1-2 micrograms/ml). The effect of in vitro treatment of human s.c. fat cells and hamster adipocytes with clonidine as well as the incidence of chronic clonidine treatment (10 days) of golden hamsters were studied by testing lipolytic and antilipolytic responses of the isolated fat cells. Moreover, binding parameters were determined on the corresponding fat cell ghosts using [3H]clonidine and [3H]yohimbine. The preincubation of hamster (90 min) and human (3 hr) fat cells with clonidine did not modify the biological responses promoted by isoproterenol or clonidine. Binding analysis showed that the number of [3H]clonidine sites was reduced by 30 to 40% in human fat cell ghosts whereas [3H]yohimbine sites were unaffected by clonidine treatment. The chronic treatment of hamsters with clonidine (10 days, 200 micrograms/day) did not influence the lipolytic and antilipolytic responses of the fat cells. However, the number of alpha-2 adrenoceptor sites, identified by [3H]clonidine was reduced (40-50%). To conclude, short-term incubations of adipocytes with clonidine or long-term in vivo treatments with this drug are without any noticeable influence on the lipolytic and antilipolytic responses of human and hamster isolated fat cells. The reduction of [3H]clonidine binding sites described in both experiments must be questioned concerning its biological relevance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester and staurosporine modulation of antagonist and agonist binding to alpha-2 adrenergic receptors: differential influence on Na+ versus guanylylnucleotide regulation.

The modulatory influence of the protein kinase C (PKC) activator phorbol dibutyrate (pDBu) and the PKC inhibitor staurosporine on the binding of the antagonist rauwolscine and the agonist (-)-epinephrine to alpha-2 adrenergic receptors was studied in plasma membranes from bovine aorta. In control membranes [3H]rauwolscine binding exhibited high (KDH = 110 pM) and low (KDL = 2.4 nM) affinity components. The addition of 0.1 mM 5'-guanylylimidodiphosphate [Gpp(NH)p] reduced binding to a single component (KD = 1.3 nM) and the addition of 140 mM NaCl increased the proportion of high affinity sites from 7 to 15%, whereas the combination of both Gpp(NH)p and NaCl did not differ from values for NaCl alone. PDBu pretreatment had little effect on [3H]rauwolscine binding with the exception of a small increase in KD in the presence of Gpp(NH)p. Staurosporine pretreatment, however, eliminated the high-affinity component in the absence of Gpp(NH)p or NaCl and rendered Gpp(NH)p ineffective. NaCl was able to restore two components of [3H]rauwolscine binding to the same extent as in untreated membranes. Epinephrine displaced [3H]rauwolscine in a biphasic manner (KDH = 93 nM, KDL = 3.5 microM; %RH = 42). In untreated membranes Gpp(NH)p reduced epinephrine affinity, but did not alter the %RH. NaCl alone increased KDL and caused a partial decrease in %RH, whereas the combination of Gpp(NH)p and NaCl was required to produce a single, low-affinity state (KD = 11.9 microM). PDBu pretreatment reduced epinephrine affinity and blocked the effectiveness of Gpp(NH)p, but the action of NaCl was more pronounced than in untreated membranes.(ABSTRACT TRUNCATED AT 250 WORDS)