Rat Myocardial Membranes Research Articles

Detection of nonopioid β‐endorphin receptor in the rat myocardium

Two selective agonists of nonopioid β-endorphin receptor, synthetic peptides TPLVTLFK (octarphin) and SLTCLVKGFY (immunorphin), were labeled with tritium to specific activity of 29 and 25 Ci/mmol, respectively. Both labeled peptides were found to bind to high-affinity naloxone-insensitive binding sites on the membranes isolated from the rat myocardium (Kd = 2.0 ± 0.2 and 2.5 ± 0.3 nM, respectively). The [(3)H]octarphin specific binding to the myocardial membranes was inhibited by unlabeled β-endorphin (Ki = 1.9 ± 0.2 nM) and immunorphin (Ki = 2.2 ± 0.3 nM). The [(3)H]immunorphin specific binding with the membranes was inhibited by unlabeled β-endorphin (Ki = 2.3 ± 0.3 nM) and octarphin (Ki = 2.4 ± 0.3 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α-endorphin, γ-endorphin, [Met(5)]enkephalin and [Leu(5)]enkephalin. Thus, β-endorphin, immunorphin and octarphin bind to the common high-affinity naloxone-insensitive receptor of the rat myocardial membranes.

Effects of Caulophine on Caffeine‐induced Cellular Injury and Calcium Homeostasis in Rat Cardiomyocytes

Caulophine is a novel fluorenone alkaloid isolated from the radix of Caulophyllum robustum Maxim. Caulophine showed high affinity for the rat myocardial cell membrane as assessed by cell membrane chromatography, suggesting that the compound may exert bioactivity in the heart. It is known that calcium plays an important role in the pathogenesis of ischaemic heart disease, and caffeine can cause calcium overload in cardiomyocytes by inducing calcium release from the sarcoplasmic reticulum. Therefore, the present study evaluated the effects of caulophine on caffeine-induced injury and calcium homeostasis in cardiomyocytes. Cardiomyocytes were pre-treated with caulophine before exposure to caffeine or potassium chloride (KCl). Cell viability was assayed using the MTT method, and lactate dehydrogenase (LDH) and malondialdehyde (MDA) were measured spectrophotometrically. Caulophine-pre-treated cardiomyocytes were incubated with Fluo-3/AM, and then caffeine or KCl was used to induce Ca(2+) overload. The total intracellular Ca(2+) concentration was measured by flow cytometry. Fluorescence densities of single cardiomyocytes were detected using a confocal microscope. Caulophine increased the viability of caffeine-injured cardiomyocytes and decreased LDH activity and MDA level in cardiomyocytes. Furthermore, caulophine significantly decreased the total intracellular free Ca(2+) concentration and intracellular calcium release in cardiomyocytes in response to caffeine. However, the same concentrations of caulophine did not affect KCl-induced calcium influx. Our results suggest that caulophine protects cardiomyocytes from caffeine-induced injury as a result of calcium antagonism. This finding provides a basis for further study and development of caulophine as a new calcium antagonist for treating ischaemic cardiovascular diseases.

U73122, an Aminosteroid Phospholipase C Inhibitor, Is a Potent Inhibitor of Cardiac Phospholipase D by a PIP2-Dependent Mechanism

The aminosteroid 1-[6-({17beta-3-methoxyestra-1,3,5(10)-trien-17-yl}amino)hexyl]-1H-pyrrole-2,5-dione (U73122) has been extensively used as a pharmacologic inhibitor of phospholipase C (PLC). The inhibitory effect of U73122 on PLC activity is most likely the result of decreased availability of phosphatidylinositol 4,5-bisphosphate (PIP2), the substrate of the PLC signal transduction pathway, rather than direct inhibition of the enzyme. PIP2 is a phospholipid with pleiotropic cellular functions, including a pivotal role in regulating cardiac phospholipase D (PLD) signal transduction. Here, we hypothesized that U73122 acts as an inhibitor of cardiac PLD activity by interference with PIP2. U73122 concentration-dependently inhibited PLD activity in rat myocardial membranes. The inhibitory effect of U73122 was significantly attenuated when assayed on solubilized PLD activity and was completely restored if solubilized PLD activity was assayed in the presence of PIP2. U73122 had no inhibitory effect on purified PLD indicating that the substance does not interact with PLD directly. These data highlight a mechanism of action of U73122 as an inhibitor of myocardial PLD by interaction with PIP2 as a cofactor for optimal PLD activity. Hence, studies using U73122 as a specific inhibitor of PLC have to take into account that PLD may be involved in some of the effects ascribed to PLC.

Reports of Death with Use of Propofol (Diprivan) for Nonprocedural (Long-term) Sedation and Literature Review

Reports of Death with Use of Propofol (Diprivan) for Nonprocedural (Long-term) Sedation and Literature Review

Propofol-induced alterations in myocardial beta-adrenoceptor binding and responsiveness.

Propofol (iv) depresses cardiovascular function in both humans and animals. However, the mechanism underlying this action has not been well described. The present study was designed to test the hypothesis that this effect of propofol results in part from an antagonism of adrenergic control of the heart. Experiments examined effects of propofol on: 1) [3H]CGP12177 (a beta-adrenoceptor antagonist) binding in rat myocardial membranes; and 2) the inotropic and chronotropic actions of isoproterenol in rat left atrial muscle and right atria, respectively. Propofol (25-200 microM) increased the apparent dissociation constant for [3H]CGP12177 without affecting binding site density. Similarly, 200 microM propofol increased the 50% effective concentration values for the dose-dependent positive chronotropic and inotropic actions of isoproterenol in right and left atria, and depressed the maximum increase in spontaneous rate elicited by this beta-adrenoceptor agonist. Other experiments demonstrated that propofol does not alter muscarinic receptor binding as monitored using [3H]quinuclidi-nylbenzilate. In conclusion, these results indicate that propofol can decrease cardiac beta-adrenoceptor responsiveness; however, the concentrations of propofol required suggest that this action contributes to the cardiovascular depression produced by this anesthetic only during large-dose bolus injection. Experiments in membranes and cardiac preparations isolated from rat heart demonstrate that relatively high concentrations of propofol (25-200 microM) are required to antagonize beta-adrenoceptor binding and tissue responsiveness.

Antioxidant effects of calcium antagonists on rat myocardial membrane lipid peroxidation.

We studied the antioxidant effects of nine calcium antagonists (nisoldipine, benidipine, nilvadipine, felodipine, nicardipine, nitrendipine, nifedipine, verapamil, and diltiazem) by means of rat myocardial membrane lipid peroxidation with a nonenzymatic active oxygen-generating system (DHF/FeCl3-ADP). The order of antioxidant potency of these agents was nilvadipine > nisoldipine > felodipine > nicardipine > verapamil > benidipine. Their IC50 values (microM) were 25.1, 28.2, 42.0, 150.0, 266.1, and 420.0, respectively. In contrast, nitrendipine, nifedipine, and diltiazem had little inhibitory effect on lipid peroxidation. These six calcium antagonists could be divided into four types on the basis of their antioxidant mechanisms. Nilvadipine, nisoldipine, and verapamil, which showed antioxidant effects both before and after the addition of active oxygen, and reduced the dihydroxyfumarate (DHF) auto-oxidation rate, were chain-breaking and preventive antioxidants. Felodipine, which showed antioxidant effects both before and after exposure to active oxygen and increased the DHF auto-oxidation rate, was only a chain-breaking antioxidant. Nicardipine, which showed an antioxidant effect only before exposure to active oxygen and reduced the DHF auto-oxidation rate, was mainly a preventive antioxidant. Benidipine, which showed an antioxidant effect only before exposure to active oxygen and had no appreciable effect on the DHF auto-oxidation rate, could interrupt the chain reaction of lipid peroxidation at the initial step alone. Although these results suggest that the antioxidant properties of some calcium antagonists may be beneficial clinically in protecting against cellular damage caused by lipid peroxidation, further studies are required to establish the antioxidant effects of these agents in vivo.

Characterization of Bradykinin B 2 Receptors in Adult Myocardium and Neonatal Rat Cardiomyocytes

Specific [125I-Tyr8]bradykinin (BK) binding was observed on myocardial membranes from adult guinea pigs, dogs, rats, and rabbits that was displaced by unlabeled BK with an IC50 between 0.1 and 30 nmol/L. In the adult guinea pig ventricular myocardium, which displays both high- and low-affinity binding, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 mumol/L) eliminated high-affinity binding and reduced total specific [2,3-prolyl-3,4-3H(N)]BK ([3H]BK) binding by > 60%. Agonist competition binding to rat myocardial membranes was characterized as being of one affinity for BK in the nanomolar range, and it was not altered by GTP gamma S. Saturation binding studies with [125I-Tyr8]BK and [3H]BK, performed on cultured neonatal rat cardiac myocytes, revealed a single class of BK binding sites with a Kd and Bmax of 0.24 +/- 0.04 nmol/L and 18.4 +/- 1.1 fmol/mg protein, respectively (approximately 1500 receptors per cell). In competitive binding assays, unlabeled BK, Hoe 140 (a specific BK B2 receptor antagonist), and des-Arg9,[Leu8]BK (a BK B1 receptor antagonist) displaced [125I-Tyr8]BK with an IC50 of 4.3, 0.041, and 307 nmol/L, respectively. In the presence of 100 mumol/L GTP gamma S, [3H]BK binding to myocyte membranes was reduced by 40%, but the IC50 did not change. Cardiac fibroblasts, evaluated in parallel to the myocytes, contain a single class of [3H]BK binding sites (248 +/- 72 fmol/mg) with a 130-fold lower relative affinity (32.4 +/- 11.3 nmol/L) than that determined in rat cardiomyocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiarrhythmic agent amiodarone possesses calcium channel blocker properties.

Amiodarone possesses multiple pharmacologic properties, including peripheral and coronary vasodilatation, negative inotropy, and negative chronotropic and dromotropic effects. These properties are shared by the group of drugs termed calcium channel blockers. We examined the interaction of amiodarone with receptors for the 1,4-dihydropyridine (DHP) calcium blockers in rat and rabbit myocardial membrane particulates. Amiodarone displaced specifically bound [3H]nitrendipine in both rat and rabbit preparations in a competitive, concentration-dependent manner at a single class of binding sites (Ki approximately 0.27 micxroM). Calcium channel activity was determined pharmacologically in a tissue bath with electrically stimulated rabbit right ventricular strips, KCl-induced aortic ring contraction, and 45Ca2+ uptake in K(+)-depolarized cultured rat cardiomyocytes. Amiodarone completely inhibited myocardial contraction (EC50 = 1.7 microM), completely antagonized depolarization-induced aortic ring contraction (EC50 = 24 nM), and significantly reduced (29% vs. control) 45Ca2+ uptake into cultured cells. The calcium channel blocking effects of amiodarone may contribute significantly to its pharmacologic profile.

Circulating antibodies against neurotransmitter receptor activities in children with congenital heart block and their mothers.

In this work we demonstrated that IgG present in the sera of patients with congenital heart block (CHB) and their mothers could bind and activate beta adrenoceptors and muscarinic cholinergic receptors of neonatal heart. These antibodies were able to inhibit in a noncompetitive manner the binding of [3H]QNB and [3H]CGP to muscarinic cholinergic and beta adrenoceptors of purified neonatal rat myocardial membranes, respectively. Moreover, IgG from children with CHB and their mothers could modify biological effects mediated by these neurotransmitter receptors, i.e., heart contractility and cAMP production. Neither binding nor biological effects were obtained using adult instead of neonatal rat atria. Both reactivities (adrenergic and cholinergic) were independent of Ro/SS-A and La/SS-B antibodies and were absent in the sera of normal women of childbearing age and of normal children. It could be concluded that antibodies against cardiac neurotransmitter receptors may be another serum factor (or factors) to be considered in the pathophysiology of the development of CHB.

Sevoflurane inhibited beta-adrenoceptor-G protein bindings in myocardial membrane in rats.

The effects of sevoflurane on the beta-adrenoceptor (beta AR) system, consisting of the receptor, stimulatory guanosine triphosphate (GTP) binding protein (Gs), and adenylate cyclase (AC), were studied in rat myocardial membrane. Cyclic adenosine monophosphate (cAMP) production rate was measured as cAMP generation stimulated by l-isoproterenol, guanosine 5"-O-(3-thiotriphosphate) (GTP gamma S), and forskolin, respectively. The receptor studies were performed by using the dihydroalprenolol ([3H]DHA) binding method. Nonspecific binding was determined in the presence of l-propranolol. beta AR binding capacity determined by the maximum number of binding sites (Bmax) and the dissociation constant (Kd) for [3H]DHA were calculated. beta AR affinity for agonists was assessed by an inhibition constant (Ki), calculated from the concentration of l-isoproterenol to produce half-maximum inhibition of [3H]DHA binding (IC50). At 2.0 mM sevoflurane, cAMP production rate was significantly depressed by 87.1% +/- 4.8% with l-isoproterenol and by 79.4% +/- 7.0% with GTP gamma S, but not significantly depressed by forskolin. In the presence of 0-2.0 mM sevoflurane, Kd for [3H]DHA and Ki for l-isoproterenol increased in a concentration-dependent manner, whereas Bmax remained unchanged. The ratio of high affinity state against the high and low affinity state of the adrenoceptor decreased significantly from 0.60 +/- 0.15 to 0.35 +/- 0.12. We conclude that sevoflurane might depress the beta AR signal transduction system by reducing ligand-receptor bindings and disrupting the relationship between the receptor and Gs.

In vivo recovery of α1-adrenoceptors in rat myocardial tissue after alkylation with phenoxybenzamine

The rate of recovery of rat myocardial α 1-adrenoceptor density and responsiveness after in vivo block with phenoxybenzamine (1 mg/kg, i.p.) have been investigated by measuring [ 3H]prazosin binding, and noradrenaline-stimulated [ 3H]inositol phosphate production. Repopulation of α 1-adrenoceptors was monoexponential, with a t 1 2 of 33 h; functional recovery was also monoexponential, with t 1 2 of 28 h. Furthermore, our results clearly demonstrate the absence of a receptor reserve for α 1-adrenoceptors mediating noradrenaline-stimulated phosphoinositide breakdown in rat myocardial tissue. These observations indicate a close relationship between the density of [ 3H]prazosin binding sites and the ability of α 1-adrenoceptors to respond to noradrenaline. Moreover, based on competition curves for inhibition of specific [ 3H]prazosin by WB-4101 to rat myocardial membranes 48 h and 7 days after the administration of phenoxybenzamine, the results suggest that rat myocardial membranes contain both α 1-adrenoceptors subtypes, i.e., α 1A and α 1B, in an approximate ratio of 20:80, and this relative ratio does not seem to be altered during the recovery process.

G-proteins in experimental hypertension: a study of spontaneously hypertensive rat myocardial and renal cortical plasma membranes.

Spontaneously hypertensive rat (SHR) myocardium is known to exhibit reduced beta-adrenergic agonist-stimulated adenylyl cyclase activity. As G-proteins play a pivotal role in transducing information from the receptor to adenylyl cyclase, a study was undertaken to assess whether changes in G-protein expression and functioning could explain this. Studies were performed in plasma membrane homogenates from vascular tissue (myocardium) from 11-week-old SHR (weighing 255 g) and control rats from both Wistar-Kyoto (WKY) and Wistar strains. Similar studies were performed in non-vascular tissue (renal cortical plasma membranes) to assess whether any observed changes are part of a more widely distributed membrane defect. G-protein function was inferred from studies of adenylyl cyclase activity and levels of G-protein subunits (Gs alpha, Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha and beta) were assessed by immunoblotting. Differences in adenylyl cyclase activity were seen in SHR compared with WKY rat myocardium: in WKY rats adenylyl cyclase activity was greater than in SHR under forskolin-stimulated conditions and in the presence of fluoride and several ligands which couple to the catalytic unit of adenylyl cyclase via Gs, including the receptor-linked species prostaglandin E1, glucagon and isoproterenol. However, with the exception of forskolin-stimulated activity, which in SHR was greater than in Wistar rats, SHR myocardial adenylyl cyclase activities were similar to those in Wistar rat membranes. Immunoblotting studies showed similar levels of G-protein subunits in all three strains. Studies of renal cortical plasma membranes failed to identify any differences in adenylyl cyclase activity or in G-protein subunit levels. SHR, WKY rats and Wistar rats exhibit differences in myocardial adenylyl cyclase activity which are not seen in renal cortical plasma membranes. These are not related to hypertension or to differences in G-protein levels, and probably reflect strain differences in Gs function.

Role of altered G-protein expression in the regulation of myocardial adenylate cyclase activity and force of contraction in spontaneous hypertensive cardiomyopathy in rats.

The question of this study was whether alterations in the inhibitory guanine-nucleotide binding protein alpha-subunits (G(i)alpha) contribute to alterations in adenylate cyclase regulation in hypertensive cardiomyopathy of spontaneously hypertensive rats (SHR). G(i)alpha was measured by pertussis toxin-catalysed 32P-adenosine 5'-pyrophosphate (ADP)-ribosylation and radioimmunochemically by competition of rat myocardial membrane extracts to DS 4 antiserum binding to the 125I-radiolabelled C terminus of retinal transducin alpha (125I-KENLKDCGLF). Cardiac beta-adrenoceptors, m-cholinoceptors as well as isoprenaline-, guanine-nucleotide [Gpp(NH)p]- and forskolin-stimulated adenylate cyclase activity and inotropic responses to isoprenaline and carbachol were studied in SHR and age-matched Wistar-Kyoto (WKY, control) rats. In native membranes of SHR there was an increase in pertussis toxin substrates, but a larger increase in the presence of non-ionic detergent Lubrol PX. The radioimmunological quantification of G(i)alpha revealed an increase in membrane extracts of SHR. In addition, myocardial beta-adrenoceptors and myocardial m-cholinoceptors were reduced in SHR compared with in WKY rats. Basal adenylate cyclase, isoprenaline-, Gpp(NH)p- and forskolin-stimulated adenylate cyclase activities were also reduced. However, in the presence of 5 mmol/l MnCl2 no differences in adenylate cyclase activities between SHR and WKY rats were detected under either condition. The present study shows that the amount of G(i)alpha-proteins and not only pertussis toxin substrates are increased in membranes of hypertrophic hearts from SHR without heart failure. The results obtained with pertussis-labelling depended strongly on the substrate quality of G(i)alpha. Increased G(i)alpha expression and reduced beta-adrenoceptor number might have functional relevance in the regulation of adenylate cyclase activity and force of contraction in SHR. An increase in G(i)alpha expression might play a pathophysiological role, not only in terminal heart failure, but also in hypertrophic cardiomyopathy.

Peroxidative damage to cardiac mitochondria: Identification and purification of modified adenine nucleotide translocase

Rat myocardial membranes exposed to free radical-generating systems exhibit both lipid peroxidation and protein alterations. The most sensitive protein, a 28-kDa polypeptide, was previously shown to increase slightly in apparent molecular weight before disappearing completely from the protein profile [ N. L. Parinandi, C. W. Zwizinski, and H. H. O. Schmid (1991) Arch. Biochem. Biophys. 289, 118–123]. We now report that isolated cardiac mitochondria contain a 28-kDa protein which responds in the same manner to treatment with Cu 2+/ t-butylhydroperoxide. The protein exhibits several characteristic properties of the mitochondrial adenine nucleotide translocase. This assignment is supported by the finding that carboxyatractyloside, a specific inhibitor of the adenine nucleotide translocase, can prevent the oxidant-induced changes in the 28-kDa protein. Efficient purification schemes for the isolation of milligram quantities of unmodified and oxidatively altered adenine nucleotide translocase from rat heart mitochondria are described.

Free radical-induced alterations of myocardial membrane proteins

Rat myocardial membranes exposed to the free radical-generating systems, Fe 2+/ascorbate, Cu 2+/ t-butylhydroperoxide, linoleic acid hydroperoxide, and soybean lipoxygenase (Type I) undergo lipid peroxidation. This is evidenced by the accumulation of thiobarbituric acid-reactive substances and the loss of both extractable phospholipids and their polyunsaturated acyl groups. Lipid peroxidation is accompanied by alterations of membrane proteins including the general loss of polypeptides and accumulation of high-molecular weight material. The most sensitive protein is a polypeptide with a molecular weight of 28 kDa. At low levels of oxidation, this protein moves incrementally to slightly higher apparent molecular weight. At higher oxidant levels or longer periods of oxidation, the protein disappears completely from the SDS-PAGE gel. The “28K reaction” occurs prior to the massive, oxidant-induced lipid alterations and may thus indicate specific adduct formation between this protein and certain peroxidized membrane phospholipids.

Prolonged administration in vivo of alpha and beta adrenergic agonists decreases insulin binding to rat myocardial membranes in vitro by different mechanisms

Male Sprague Dawley rats were continuously treated in vivo for 6, 12 and 20 hours with a combination of an alpha- (beta-) adrenoreceptor agonist and a beta- (alpha-) adrenoreceptor antagonist in subcutaneously implanted depot tablets. Crude membranes prepared from myocardial cells exhibited a decreased maximum binding of [ 125I]-insulin after 20 hours irrespective of the treatment applied. Scatchard and non-linear regression analysis of the displacement curves assuming two non-cooperative binding sites revealed a downregulation of the high affinity receptors for about 85% and a concomitant 2.5-fold increased receptor affinity under beta-adrenergic influence. In contrast, alpha-adrenergic treatment did not affect the receptor number but decreased the high affinity by 70%. The low affinity binding sites were virtually unaffected by the different treatments. The phospholipid and cholesterol contents of the membranes were not significantly altered. The phospholipid/cholesterol ratios after 12 and 20 hours of alpha-adrenergic treatment, however, were decreased. We suggest that the decreased binding activity of insulin receptors on rat myocardial membranes after continuous in vivo treatment with alpha- and beta- adrenergic agonists is mediated by different mechanisms.

Developmental expression of dystrophin on the rat myocardial cell membrane

The Duchenne muscular dystrophy gene product, dystrophin, is expressed on the cell membrane of skeletal and cardiac muscles. We examined the developmental time course of dystrophin expression in the rat ventricular myocardium. Dystrophin was immunohistochemically undetectable on the 15th embryonic day, but a small amount was expressed on the cell membrane on the 17th embryonic day. The amount increased during the perinatal period reaching adult level at 2 weeks after birth. These findings were supported by immunoblot analysis. Chemical sympathectomy in newborn rats by 6-hydroxydopamine (prevention of innervation) had no detectable influence on myocardial dystrophin expression at 2 weeks after birth. Our results show that dystrophin lining on the rat myocardial cell membrane increases during the perinatal period, and that this process is little influenced by the development of sympathetic innervation.

Decrease in rat cardiac beta 1- and beta 2-adrenoceptors by training and endurance exercise

The cardiac β-adrenoceptor adaptation to physical activity was investigated in rats which were subjected to a six-week endurance swimming training (ET;n=7) and a training of high intensity (MT; n=7). In addition, the effect of a single bout of endurance exercise without preceding training (EE; n=7) was evaluated. These groups were compared with a sedentary control group (C; n=9). Beta-adrenergic receptors in rat myocardial membranes were labelled using the high affinity antagonist radioligand (−) 125iodocyanopindolol (ICYP). Computer modelling techniques provided estimates of the maximal binding capacity (B max) and the dissociation constants (K D). Tissue was constantly kept at temperatures of ≤4°C and incubated at 4°C for 18 h in buffer containing 100 μM GTP so as to prevent masking of β-adrenoceptors by endogenous norepinephrine. In comparison with the C group (B max=43.2±1.6 fmol/mg protein, K D=11.7±1.5 pM) computerized coanalyses of saturation binding data of ET, MT, and EE revealed a 13.0 %, 25.5 % and 16.6 % decrease in B max (P<0.01), respectively, without significantly differing K D values (10.6 pM, 9.0 pM, 10.5 pM, respectively). We provide the first evidence that acute exercise lowers the sarcolemmal β-adrenoceptor number in the rat heart. In the competition radioligand binding, CGP20712A and ICI118.551 were employed as subtype-selective antagonists of β 1- and β 2-adrenoceptors, respectively, to determine the relative proportions of the receptor subtypes. The ratio of β 1- β 2 - adrenoceptors in C was 67.5:32.5 and no statistically significant variation occured in animals subjected to physical activity. On the basis of published data we assume that acute exercise induces a sequestration of β-adrenoceptors from the cell surface to some intracellular compartment, whereas the molecular basis of the chronic β-adrenoceptor down-regulation may involve a training-induced reduction in receptor synthesis. Our findings on cardiac β-adrenoceptor adaptation to physical activity may represent one of the mechanisms underlying the relative bradycardia in trained subjects

Binding characteristics of 3H-CGP12177 to .BETA.-adrenoceptors in rat myocardial membranes.

The present study was designed to examine the selectivity of 3H-CGP-12177 (4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazole-2-one hydrochloride) for beta 1- and beta 2-adrenergic receptors by the Scatchard and the displacement analysis. The plots of specific binding obtained from the Scatchard analysis using 3H-CGP12177 for the rat myocardium membrane were uniphasic when the non-specific binding was determined by the use of 10 microM I-propranolol, and the Kd and Bmax values were 408.53 +/- 67.20 pM and 12.27 +/- 0.83 fmoles/mg protein, respectively. On the other hand, two binding sites were observed in the displacement curve when I-metoprolol was used as a competitor. The existence of these two binding sites implied the selectivity of 3H-CGP12177 to beta-adrenoceptors because 3H-CGP12177 was 1.8-fold more selective towards beta 1-adrenoceptors than beta 2-adrenoceptors. In addition, these two binding sites could be regarded as beta 1- and beta 2-adrenergic receptors from the evaluation of the binding characteristics beta-adrenoceptors by the displacement analysis using beta-selective antagonists. Thus, 3H-CGP12177, a hydrophilic radioligand, was useful for the binding assay of beta-adrenoceptors in rat myocardial membranes.

Binding Characteristics of 3H-CGP12177 to β-Adrenoceptors in Rat Myocardial Membranes

The present study was designed to examine the selectivity of 3H-CGP-12177 (4-(3-t-butylamino-2-hydroxyproppxy)-[5,7-3H]benzimidazole-2-one hydrochloride) for β1- and β2-adrenergic receptors by the Scatchard and the displacement analysis. The plots of specific binding obtained from the Scatchard analysis using 3H-CGP12177 for the rat myocardium membrane were uniphasic when the non-specific binding was detemined by the use of 10 μM l-propranolol, and the Kd and Bmax values were 408.53±67.20 pM and 12.27±0.83 fmoles/mg protein, respectively. On the other hand, two binding sites were observed in the displacement curve when l-metoprolol was used as a competitor. The existence of these two binding sites implied the selectivity of 3H-CGP12177 to β-adrenoceptors because 3H-CGP12177 was 1.8-fold more selective towards β1-adrenoceptors than β2-adrenoceptors. In addition, these two binding sites could be regarded as β1 and β2-adrenergic receptors from the evaluation of the binding characteristics of β-adrenoceptors by the displacement analysis using β-selective antagonists. Thus, 3H-CGP12177, a hydrophilic radioligand, was useful for the binding assay of β-adrenoceptors in rat myocardial membranes.