Binding of catecholamines to alpha-1 acid glycoprotein, albumin and lipoproteins in human serum

The binding of catecholamines in human serum was determined by equilibrium dialysis at 37 degrees. For serum concentrations of 10-15 nM the bound fractions were 28.8 +/- 2.2%, 25.7 +/- 1.7% and 22.2 +/- 2.2% for (+/-)-isoproterenol (IPR), (+/-)-norepinephrine (NE) and (+/-)-epinephrine (EPI), respectively. At higher serum concentrations saturation occurred. Alpha-1 acid glycoprotein (AAG) possessed one high affinity binding site and approximately 10 low affinity sites. The catecholamines were bound to AAG with the same order of potency for both classes of binding sites: IPR (Kd1: 100 microM Kd2: 2.2 mM) greater than NE (Kd1: 120 microM, Kd2: 6.5 mM) greater than EPI (Kd1: 140 microM, Kd2: 14 mM). Human serum albumin (HSA) and lipoproteins (SLP) interacted with the catecholamines in a non-saturable manner. IPR showed the strongest and EPI the weakest association to both of these serum protein fractions. (-)-Propranolol was able to inhibit the binding of IPR in serum and to isolated AAG, but not to HSA or to SLP. The present results show that AAG is an important catecholamine-binding protein in human serum. AAG, but not HSA or SLP, possesses binding sites shared by adrenergic receptor stimulators and blockers.     

Beta-adrenoceptors in human alveolar macrophages isolated by elutriation.

1. beta-adrenoceptors on human alveolar macrophages obtained by bronchoalveolar lavage (BAL) from healthy smoking volunteers (n = 26) were characterized by studying cyclic AMP (cAMP) accumulation in intact macrophages evoked by adrenaline or isoprenaline, with or without appropriate antagonists and by radioligand binding to macrophage membranes, using [125I]-iodopindolol (125IPIN) as beta-adrenoceptor ligand. 2. In a second study, cAMP responses of alveolar macrophages to isoprenaline and PGE1 and of peripheral blood lymphocytes to isoprenaline were compared in smoking and non-smoking healthy volunteers (n = 9 + 9), as our initial studies were performed in smokers, due to their higher cell yield. 3. BAL yielded 47 +/- 23 x 10(6) cells in smokers and 12 +/- 6 x 10(6) cells in non-smokers with a recovery of 82 +/- 8% in the elutriation step (means +/- s.d.). The cell preparation consisted of 99.2 +/- 0.8% macrophages and their viability (trypan blue exclusion) was 97.5 +/- 5.2%. 4. Isoprenaline or adrenaline increased cAMP accumulation approximately 40-fold with or without the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 10(-4) M), which enhanced basal and stimulated cAMP accumulation approximately five-fold. Peak responses were seen after 2 min. EC50s for isoprenaline and adrenaline were 3-5 x 10(-7) M. Phentolamine did not alter responses to adrenaline, indicating absence of inhibitory alpha 2-adrenoceptors. Propranolol inhibited isoprenaline induced cAMP accumulation stereoselectively; pD2-values were 8.2 for (-)-propranolol, 5.6 for atenolol and 7.5 for ICI 118,551, suggesting a predominance of beta 2-adrenoceptors. 5. Specific 125IPIN binding to macrophage membranes was rapid and saturable. Non-specific binding was determined in the presence of 1 microM (-)-propranolol. KD values were 71 +/- 7 pM and the density of specific binding sites was 36 +/- 3 fmol mg-1 protein (three experiments on a membrane pool from 10 subjects; r values for Scatchard analyses = 0.98 +/- 0.01). Similar values were obtained when 200 microM isoprenaline (+ GTP) was used to assess non-specific binding. Competition experiments again showed stereoselectivity for propranolol and a predominance of beta 2-adrenoceptors, as judged by the displacement of specific 125IPIN binding by atenolol and ICI 118,551. 6. Macrophages from smokers responded with less marked cAMP accumulation upon stimulation with isoprenaline or PGE1 than did cells from non-smokers (difference approximately 30%; P less than 0.05 for both agonists) in the presence of IBMX. Thus macrophages from smokers may produce less cAMP due to post-receptor changes in responsiveness.(ABSTRACT TRUNCATED AT 400 WORDS)     

Guanine nucleotides regulate both agonist and antagonist binding to cod brain alpha 1-adrenoceptors

The effect of guanine nucleotides on the binding of 3H-prazosin and its displacement by various agonists and antagonists were studied in membranes prepared from the cod brain. When cod brain membranes were maintained in a buffer containing 8 mM Mg2+, GTP (1 mM) was found to increase the specific binding of 3H-prazosin Computer modelling 3H-prazosin saturation curves, suggested that the number of binding sites for 3H-prazosin was increased by 18 +/- 5% by GTP without affecting the affinity for 3H-prazosin (dissociation constant, Kd, 56 +/- 3 pM). Displacement of 3H-prazosin by adrenaline produced shallow displacement curves. Computer modelling these curves suggested that adrenaline bound to two sites - one high and one low affinity site - the Kd's being 0.14 +/- 0.03 (KH) and 7.6 +/- 0.5 microM (KL), respectively. When 1 mM GTP was present the displacement curves were shifted to the right and became steeper. Computer modelling these curves suggested that adrenaline now bound to only one low affinity site with a Kd of 7.6 +/- 0.5 microM. When unlabelled prazosin was used to displace 3H-prazosin the displacement curves were uniphasic and steep, irrespective of whether GTP was present or not. Computer modelling these curves suggested that prazosin bound to only one site with a Kd of 68 +/- 11 pM. In the absence of Mg2+ and in the presence of EDTA (0.8 mM) the displacement curve of adrenaline became steeper and the effect of GTP was almost abolished. During these conditions the ability of GTP to enhance 3H-prazosin binding was also abolished.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of serum, alpha-1 acid glycoprotein, lipoproteins and albumin on human mononuclear leucocyte beta-adrenoceptors

The effects of serum, alpha-1 acid glycoprotein (AAG), serum lipoproteins (SLP) and human serum albumin (HSA) on 3H-(-)-dihydroalprenolol (3H-(-)-DHA) binding and (-)-isoproterenol ((-)-IPR) induced cyclic AMP (cAMP) elevation in human peripheral blood mononuclear leucocytes (MNL) were investigated. The saturable binding of 3H-(-)-DHA was decomposed into two classes of binding sites with maximum binding capacity of approximately 1400 and 30000 sites/cell and with dissociation constants (Kd) of approximately 0.7 and 65 nM. Stimulation of the MNL beta-adrenoceptors by (-)-IPR caused a concentration dependent cAMP accumulation (EC50 approximately 0.2 microM) with maximum level approximately 250% above basal. For all single leucocyte preparations, 30-35 min. exposure to serum, AAG and SLP increased the number of beta-adrenoceptors with 100-200% and the maximal responsiveness to (-)-IPR with 30-90%. The presence of proteins did not change the Kd or the EC50. (-)-Alprenolol inhibited concentration dependently the serum induced increment in (-)-IPR-responsiveness. Serum, AAG and SLP did also increase the number of low affinity binding sites with 25-40% without effect on the Kd. HSA had no consistent effect on beta-adrenergic binding or stimulation. The present study shows that serum, AAG and SLP influence the number and function of MNL beta-adrenoceptors in vitro.     

Inhibitory effects of catecholamines on cholinergically and non-cholinergically mediated contractions of guinea-pig isolated bronchial muscle

The actions of catecholamines on the responses evoked by electrical field stimulation or by acetylcholine and substance P in guinea-pig bronchial strip chain have been examined. Electrical field stimulation evoked a biphasic contraction, consisting of a cholinergically-mediated fast contraction followed by a non-cholinergically-mediated slow contraction. All catecholamines tested caused a concentration-dependent reduction in the height of the biphasic contraction, where non-cholinergic contractions were more potently inhibited. The inhibitory effect of isoprenaline was largely prevented by propranolol (2 microM) alone, whereas those of noradrenaline and adrenaline were prevented by treatment with both propranolol (2 microM) and yohimbine (2 microM). The inhibitory effect of dopamine was unaffected either by propranolol (2 microM), yohimbine (2 microM) or haloperidol (10 microM). Submaximal contractions of bronchial muscle evoked by exogenous acetylcholine (2 microM) or substance P (0.2 microM) were also inhibited by catecholamines, except dopamine, but the effects were antagonized by propranolol (2 microM) alone. The results suggest that in guinea-pig isolated bronchial muscle, catecholamines can inhibit both cholinergic and non-cholinergic excitatory neurotransmissions not only by postjunctional beta-adrenoceptors but also by prejunctional alpha 2-adrenoceptors.     

Characterization of alpha adrenoceptors in pial arteries of the bovine brain

When attempting to characterize the nature of adrenoceptors in bovine pial arteries, we found specific 3H-yohimbine binding was saturable, reversible and of high affinity (KD = 18.3 +/- 1.2 nM) with a Bmax of 687 +/- 27 fmol/mg protein (N = 4). On the other hand, there was no specific 3H-prazosin binding in these tissues. Scatchard and Hill plot analyses of specific 3H-yohimbine binding indicated one class of binding sites. From kinetic analyses of the data, association and dissociation rate constants of 1.6 +/- 0.3 X 10(7) M-1min-1 and 0.51 +/- 0.04 min-1, respectively, were calculated (N = 3). The dissociation constant from the equation KD = K-1/K+1 was 35.7 +/- 7.6 nM, such being in good agreement with the KD value estimated from Scatchard plots. Specific binding of 3H-yohimbine was displaced effectively by alpha 2 adrenergic agents and less effectively by alpha 1 adrenergic agents or beta adrenergic agents. Ki values for adrenergic drugs of 3H-yohimbine binding were as follows: yohimbine, 25 nM; clonidine, 260 nM; methoxamine, 6.8 microM; propranolol, 8.7 microM; prazosin, 21 microM; phenylephrine, 22 microM; noradrenaline, 27 microM; adrenaline, 66 microM; isoproterenol, 3,300 microM. These results indicate that alpha adrenoceptors in the bovine cerebral arteries can be classified as the alpha 2 subtype.     

The mechanism of the inhibitory action of adrenaline on transmitter release in bullfrog sympathetic ganglia: independence of cyclic AMP and calcium ions.

The effects of adrenaline and dibutyryl adenosine 3':5' - cyclic monophosphate (db cyclic AMP) on nicotinic transmission in bullfrog sympathetic ganglia were compared by use of an intracellular recording technique. The evoked release of transmitter, acetylcholine (ACh), was decreased in the presence of adrenaline (10-100 microM), while the postsynaptic sensitivity to ACh was unchanged (10 microM adrenaline) or slightly reduced (100 microM). Transmitter release was similarly inhibited by dopamine (10 microM), but not by isoprenaline (10 microM). The inhibitory action of adrenaline on transmitter release was blocked by phenoxybenzamine but not by propranolol. The inhibition of transmitter release was independent of the external calcium concentration. The evoked release of transmitter and the electrical properties of the postsynaptic membrane were unchanged during exposure to db cyclic AMP (1-4 mM), while the postsynaptic sensitivity to ACh was slightly but significantly depressed. The spontaneous release of transmitter in a high K+ (10 mM) solution was decreased in the presence of adrenaline (100-300 microM), but unchanged with db cyclic AMP (4 mM). In contrast to the effects during exposure, both the evoked and spontaneous release of transmitter were enhanced after the removal of adrenaline or db cyclic AMP. Neither adrenaline (100 microM) nor db cyclic AMP (4 mM) affected the presynaptic spike and synaptic delay. It is concluded that adrenaline mainly inhibits the release of ACh from the presynaptic terminals through its alpha-action, while db cyclic AMP reduces slightly the postsynaptic sensitivity to ACh and that both agents facilitate transmitter release when they are removed from the presynaptic terminals. It is further suggested that the inhibitory action of adrenaline is independent of endogenous cyclic AMP and calcium ions.     

Inhibitory effects of sympathomimetic drugs on cholinergically mediated contractions of guinea-pig isolated tracheal muscle

The experiments examine the actions of sympathomimetic drugs on the responses evoked by electrical field stimulation or by acetylcholine in guinea-pig tracheal strip chains. Electrical field stimulation evoked contractions which were cholinergically mediated, in the presence of guanethidine (10 microM) and indomethacin (2 microM). All the sympathomimetic drugs tested caused a concentration-dependent reduction in the height of these contractions. Inhibitory effects of isoprenaline and terbutaline were largely prevented by propranolol (2 microM) alone, whereas those of clonidine, oxymetazoline, lidamidine and WHR1370 were prevented by yohimbine alone (2 microM). Treatments with both propranolol and yohimbine were required to prevent the inhibitory effects of noradrenaline, adrenaline and dopamine. Contractions evoked by exogenous acetylcholine (0.1-3 microM) were also inhibited by all catecholamines and terbutaline, but not by clonidine, oxymetazoline, lidamidine and WHR1370. The inhibitory effects were antagonized by propranolol (2 microM) alone. The results suggest that in guinea-pig isolated tracheal muscle, sympathomimetic drugs can inhibit cholinergic neurotransmission not only by postjunctional beta 2-adrenoceptors but also by prejunctional alpha 2-adrenoceptors.     

Use of 1-anilino-8-naphthalene sulfonate as a fluorescent probe in the investigation of drug interactions with human alpha-1-acid glycoprotein and serum albumin

We report a rapid method for the characterization of the human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) interactions with drugs. The binding of 1-anilino-8-naphthalene sulfonate (ANS) to AAG and HSA was measured by fluorescence spectroscopy. Fluorescence data indicated that ANS was bound tightly to at least one site on AAG, with an affinity constant of 1.35 x 10(6) M-1. The fluorescence of an ANS:AAG complex was quenched by the binding of various drugs. Fluorescence quenching of the HSA:ANS complex showed a single site with an affinity constant of 0.72 x 10(6) M-1. The interaction of AAG and HSA with ANS or other drugs was also studied by comparative equilibrium dialysis. [14C]Pipequaline was used as an AAG and HSA site marker. [14C]Pipequaline seems to share sites I (azapropazone) and II (diazepam and ibuprofen) of HSA. However, high concentrations of warfarin were unable to displace [14C]pipequaline. On the other hand, it was shown that palmitic acid decreased, whereas bilirubin increased the pipequaline binding.     

Adrenoceptors mediating relaxation to catecholamines in rat isolated jejunum.

1. The characteristics of adrenoceptors mediating relaxation to catecholamines in rat isolated jejunum were investigated. 2. Catecholamines and BRL 37344 produced relaxation of the KCl-contracted strips with an order of potency of isoprenaline (1.0) > BRL 37344 (0.63) > noradrenaline (0.1) > adrenaline (0.04). 3. In the presence of both prazosin (1 microM) and propranolol (1 microM) only small dextral shifts of the concentration-response curves to agonists were observed and an order of potency of BRL 37344 (2.5) > isoprenaline (1.0) > noradrenaline (0.2) > adrenaline (0.1) was obtained. 4. In the presence of prazosin (1 microM) and propranolol (1 microM), cyanopindolol (0.1-10 microM) produced a concentration-dependent rightward shift of the concentration-response curve to adrenaline with a Schild slope not significantly different from unity and a mean pA2 value of 7.01. 5. The resistance of relaxant responses to propranolol, the relatively high potency of BRL 37344 compared to catecholamines and the competitive antagonism of relaxant responses to adrenaline by cyanopindolol suggest that beta-adrenoceptors in rat small intestine are mainly atypical in nature.     

Contractions mediated by alpha 1-adrenoceptors and P2-purinoceptors in a cat colon circular muscle.

1. The postjunctional excitatory and inhibitory effects of adrenoceptor and purinoceptor agonists and antagonists were studied in circular smooth muscle strips of cat colon. 2. In the presence of tetrodotoxin (0.5 microM), noradrenaline caused contraction or relaxation of circular smooth muscle at resting tension or with raised tone, respectively. The noradrenaline-evoked contractions were potentiated and the noradrenaline-evoked relaxations were antagonized by propranolol (1 microM), suggesting beta-adrenoceptor involvement. 3. At resting tension, noradrenaline, adrenaline and the selective alpha 1-adrenoceptor agonist, phenylephrine, caused concentration-dependent contractile responses, with EC50 values of 1.8 +/- 0.2 microM, 1.9 +/- 0.4 microM and 4.3 +/- 1.7 microM, respectively. The EC50 values and the amplitude of maximal responses were not significantly different from one another. Clonidine (0.1-500 microM), a selective alpha 2-adrenoceptor agonist, was not effective. 4. Prazosin (0.1-9 microM), competitively antagonized the contractile effects of noradrenaline with an estimated pA2 value of 6.93 and a slope of 1.07 +/- 0.03. The Kb values, estimated from a single shift (0.1 microM prazosin) of the concentration-response curves to noradrenaline, adrenaline and phenylephrine were 92.8 +/- 9.3 nM, 108.7 +/- 6.4 nM and 18.4 +/- 3.1 nM, respectively. 5. At resting tension, adenosine 5' triphosphate (ATP, 5-1000 microM), alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP, 0.05-50 microM), beta,gamma-methylene adenosine 5'-triphosphate (beta,gamma-MeATP, 0.5-100 microM), and 2-methylthioadenosine 5'-triphosphate (2-MeSATP, 1-500 microM) caused concentration-dependent contractions with EC50 values of 60.5 +/- 15.9 microM, 0.7 +/- 0.1 microM, 7.6 +/- 0.1 microM and 25.3 +/- 12.8 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of alpha-1-acid glycoprotein administration on propranolol binding and beta blockade in rats

Alpha-1-acid glycoprotein (AAG), 750 mg/kg, was administered to rats to determine its effect on propranolol binding and beta blockade. Anesthetized rats received [3H]propranolol i.v., followed in 15 min by human AAG or bovine serum albumin, 750 mg/kg. AAG treatment produced a human AAG concentration in serum of 7.76 +/- 1.17 mg/ml, several times higher than the endogenous serum AAG concentration in stressed rats. AAG treatment significantly increased the heart rate response to isoproterenol, compared to albumin (95.4 +/- 19.6 vs 28.3 +/- 16.7% of baseline value, measured 45 min after propranolol, P less than 0.001). AAG-treated rats had greater [3H]propranolol binding in serum (93.0 +/- 3.2 vs 76.7 +/- 3.0%, P less than 0.01) and a lower calculated unbound [3H]propranolol concentration in serum (2.7 +/- 1.3 vs 7.4 +/- 3.1 X 10(6) dpm/ml, P less than 0.001) than albumin-treated rats. These data demonstrate that AAG can alter propranolol pharmacokinetics and pharmacodynamics even when administered after the propranolol effect is evident. Because the reported affinity of propranolol for cardiac beta receptors is 10,000 times greater than its affinity for AAG, these data suggest that AAG acted by altering propranolol disposition rather than by directly competing with beta receptors for drug.     

Alpha 1-acid glycoprotein reverses cocaine-induced sodium channel blockade in cardiac myocytes

Alpha 1-acid glycoprotein (AAG) is an acute phase protein capable of binding basic drugs. This action explains its reversal of sodium channel blockade by drugs such as amitriptyline and quinidine. We report here the reversal of cocaine-induced sodium channel blockade by AAG. The sodium channel blocking property of cocaine is a major mechanism behind cocaine-induced sudden cardiac death, since sodium channels play a key role in the initiation and regulation of the heart beat. Voltage-gated sodium current (I(Na)) was recorded using whole-cell patch-clamp techniques. Guinea-pig cardiac ventricular myocytes were isolated and continuously perfused at room temperature with physiological solutions. At concentrations ranging from 5 to 320 microM cocaine showed a dose-dependent and reversible blockade of I(Na) with an IC50 of 45.9 microM. The addition of equimolar amounts of AAG to cocaine produced almost complete reversal of cocaine's effects, suggesting a single binding site for cocaine on the AAG molecule. With changes of peak I(Na) normalized against control as 1, cocaine at 20 and 40 microM reduced I(Na) to 0.62+/-0.042 (n = 6) and 0.57+/-0.052 (n = 9), respectively, and the addition of an equimolar concentration of AAG reversed I(Na) to 0.86+/-0.022 and 0.91+/-0.060, respectively. In conclusion: AAG reverses cocaine-induced sodium channel blockade in a dose-dependent manner, indicating a therapeutic potential to reverse acute cocaine cardiac toxicity.     

Study of beta-adrenoceptors and beta-adrenergic responsiveness in cultured "preneoplastic-like" and neoplastic rat hepatocytes

The presence of specific binding sites for tritiated CGP-12177, a beta-adrenergic antagonist, was investigated in the preneoplastic-like C1I cell-line and in Morris hepatoma MH3924 cells. It was found that C1I cells possess beta-adrenoceptors with the following characteristics: KD = 1.58 +/- 0.56 nM and Bmax = 4.41 +/- 0.88 fmol/10(6) cells. No specific binding sites could be found on MH3924 cells. Stimulation of the C1I cells beta-adrenoceptors by isoprenaline, salbutamol, adrenaline and noradrenaline induced cyclic AMP accumulation. Noradrenaline was, however, a hundred times less efficient than adrenaline, as is the case in normal rat hepatocytes. The order of potency of beta-antagonists either to displace the bound radioligand or to counteract isoprenaline induced cyclic AMP accumulation (IPS-339 greater than propranolol much greater than atenolol) indicates that the adrenoceptors present on the C1I cells are of the beta 2-subtype.     

Analysis of the hyperpolarizing effect of catecholamines on canine cardiac Purkinje fibres.

1. The hyperpolarization induced by catecholamines on barium-depolarized (0.2-0.8 mM BaCl) canine cardiac Purkinje fibres, in vitro, was studied by use of conventional microelectrode recordings of transmembrane electrical potentials. 2. Noradrenaline, adrenaline and isoprenaline hyperpolarized Purkinje fibres in a concentration-dependent manner from a threshold concentration around 5 nM. The three catecholamines were shown to be approximately equipotent. Tachyphylaxis was observed when the interval between catecholamine applications was less than 15 min. 3. Atenolol (10 microM) blocked the hyperpolarization reversibly and theophylline (0.5 mM) potentiated it. 4. Tetrodotoxin (5 microM) did not affect the hyperpolarization induced by isoprenaline. Acetylcholine and histamine, up to 10 microM, were not effective in hyperpolarizing Purkinje fibres. 5. Low extracellular potassium concentrations (zero and 1 mM) did not affect the hyperpolarization, but high extracellular potassium concentrations (10-20 mM), markedly reduced the effect of isoprenaline (100 nM). 6. Reduction of the extracellular sodium concentration produced a roughly proportional reduction in the isoprenaline-induced hyperpolarization. The hyperpolarization was reversibly blocked in 34 mM sodium Tris-Tyrode solution. 7. The hyperpolarization was not reduced in Tyrode solution containing 0.6 mM calcium, but was drastically reduced in zero-calcium Tyrode solution. This effect was reversible. 8. Addition of verapamil (5-10 microM) diminished the hyperpolarization, in a concentration-dependent manner. This effect was partially reversed after washing. 9. Ouabain (0.7-1 microM) significantly reduced the isoprenaline-induced hyperpolarization, but 2,4-dinitrophenol (0.2 mM) did not affect it. 10.Caesium chloride (20 mM) abolished the hyperpolarization. The blockade was only partially reversed upon washing.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Beta 1-, beta 2- and atypical beta-adrenoceptor-mediated relaxation in rat isolated aorta

beta-adrenoceptor-mediated relaxation was investigated in ring preparations of rat isolated thoracic aorta. Rings were pre-constricted with a sub-maximal concentration of noradrenaline (1 microM) and relaxant responses to cumulative concentrations of beta-adrenoceptor agonists obtained. The concentration-response curve (CRC) to isoprenaline was shifted to the right by propranolol (0.3 microM) with a steepening of the slope. Estimation of the magnitude of the shift from EC(50) values gave a pA(2) of 7.6. Selective beta(1)- and beta(2)-adrenoceptor antagonists, CGP 20712A (0.1 microM) and ICI 118551 (0.1 microM), respectively, produced 4 and 14 fold shifts of the isoprenaline CRC. Atypical beta-adrenoceptor agonists also produced concentration-dependent relaxation of aortic rings. The order of potency of the beta-adrenoceptor agonists was (-log EC(50)): isoprenaline (6. 25)>cyanopindolol (5.59)>isoprenaline+propranolol (5.11)>CGP 12177A (4.40)>ZD 2079 (4.24)>ZM 215001 (4.07)>BRL 37344 (3.89). Relaxation to CGP 12177A and ZM 215001 was unaffected by propranolol (0.3 microM). SR 59230A (相似文献   

Characterization and localization of atypical beta-adrenoceptors in rat ileum.

1. Homogenate binding studies and receptor autoradiography have been used to examine the binding characteristics and localization of propranolol-resistant (-)-[125I]-cyanopindolol (CYP) binding sites in rat ileum. 2. Saturation studies with (-)-[125I]-CYP and homogenates of rat ileum identified a site with pKD 8.89 +/- 0.08 and Bmax = 50.3 +/- 4.1 fmol mg-1 protein (n = 6). Both beta 1- and beta 2-adrenoceptors (AR) were not detected in these preparations. 3. (-)-Isoprenaline infusion (400 micrograms kg-1 h-1) for 14 days caused no significant change in the density of (-)-[125I]-CYP binding which was 48.9 +/- 12.8 and 40.6 +/- 12.3 fmol mg-1 protein in control and isoprenaline-treated animals respectively (n = 6) (P = 0.97). 4. Competition for (-)-[125I]-CYP binding in the presence of 0.1 microM (-)-propranolol gave affinity values for CYP, tertatolol, alprenolol, ICI 118551 and CGP 20712A that correspond to known affinities at atypical beta-ARs. Stereoselectivity ratios for tertatolol and alprenolol were low. 5. Autoradiographic localization of propranolol resistant (-)-[125I]-CYP binding showed sites associated with the mucosa and to a lesser extent to the muscularis. A small population of beta 2-ARs were detected located predominantly in the longitudinal and circular smooth muscle layers. 6. This study identifies an (-)-[125I]-CYP binding site in rat ileum that is resistant to blockade by propranolol (0.1 microM), is located predominantly in the mucosa, shows resistance to downregulation by isoprenaline and has binding characteristics of the atypical beta-AR.     

Increase in calcium channel current by beta-adrenoceptor agonists in single smooth muscle cells isolated from porcine coronary artery.

1. The action of catecholamines (isoprenaline and noradrenaline) and forskolin on membrane currents was studied in single cells freshly dispersed from the pig coronary artery by use of the whole-cell clamp method, usually with electrodes containing CsCl. 2. In normal Krebs solution, with and without 30 mM tetraethylammonium (TEA) and 0.5 mM 4-aminopyridine, isoprenaline (1-5 microM) clearly increased the inward currents elicited by membrane depolarization, without affecting the holding current at -80 mV. The same effect was observed when the external Cl- was replaced with isethionate. The outward current recorded with K(+)-containing electrodes was not significantly affected by isoprenaline. 3. In the presence of 67 mM Ba2+ and 30 mM TEA, the maximum inward current recorded with CsCl containing electrodes was 119 +/- 7 pA (the mean +/- s.e.mean, n = 90) in cells where the current was larger than 30 pA. The L-type Ca2+ channel was considered to be responsible for these currents, based on the threshold voltage, the slow time course of decay, the large depolarization necessary to produce inactivation, and the high susceptibility to the Ca2+ channel antagonist, nicardipine. 4. Isoprenaline and noradrenaline increased the amplitude of inward currents evoked by depolarizing pulses. The maximum inward current was potentiated by 43 +/- 7% (n = 12) by isoprenaline and 39 +/- 10% by noradrenaline (n = 6) at a concentration of 1 microM. These effects were strongly inhibited by propranolol, but not phentolamine. Forskolin (10 microM) also potentiated the currents to a similar degree. 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of prazosin to alpha 1-acid glycoprotein and albumin: effect of protein purity and concentrations

Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93-95% was observed at 37 degrees for therapeutic drug concentrations. Both alpha 1-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd approximately 0.8 microM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd approximately 30 microM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd approximately 8 microM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.