Decrease or increase in cardiac muscarinic cholinergic receptor number in rats treated with methacholine or atropine

The effects of chronic treatment of the rat with methacholine and atropine on the cardiac muscarinic cholinergic receptors were investigated. [³H]Quinuclidinyl benzilate ([³H]QNB) was used to directly estimate the number and affinity of the receptors in the heart ventricular membrane. Methacholine treatment decreased, in a dose-related and time-dependent manner, the specific binding of [³H]QNB by 34% as compared to the control. Atropine treatment, on the other hand, resulted in a dose-related increase (28 to 66%) in the number of the receptors. The equilibrium dissociation constant (K_D) of the receptors for the ligand was the same (about 200 pM) for the control and the methacholine treated groups of rats, whereas a dose-related increase (39 to 105%) in the K_D was noted for the atropine treated rats. Similarly, the concentration of acetylcholine causing a 50 percent inhibition (IC₅₀) of the [³H]QNB binding was unaltered for the methacholine treated rats (4 μM), but it was increased 43% for the atropine treated rats.     

Characterization of muscarinic acetylcholine receptors in the avian salt gland

Electrolyte and fluid secretion by the avian salt gland is regulated by activation of muscarinic acetylcholine receptors (R). In this study, these receptors were characterized and quantitated in homogenates of salt gland from domestic ducks adapted to conditions of low (freshwater, FW) and high (saltwater, SW) salt stress using the cholinergic antagonist [3H]-quinuclidinyl benzilate (QNB). Specific binding of the antagonist to receptors in both FW- and SW-adapted glands reveals a single population of high affinity binding sites (KdFW = 40.1 +/- 3.0 pM; KdSW = 35.1 +/- 2.1 pM). Binding is saturable; RLmaxFW = 1.73 +/- 0.10 fmol/micrograms DNA; RLmaxSW = 4.16 +/- 0.31 fmol/micrograms DNA (where L is [3H]QNB and RL the high affinity complex). Calculated average cellular receptor populations of 5,800 sites/cell in FW-adapted glands and 14,100 sites/cell in SW-adapted glands demonstrate that upward regulation of acetylcholine receptors in the secretory epithelium follows chronic salt stress. The receptor exhibits typical pharmacological specificities for muscarinic cholinergic antagonists (QNB, atropine, scopolamine) and agonists (oxotremorine, methacholine, carbachol). In addition, the loop diuretic furosemide, which interferes with ion transport processes in the salt gland, competitively inhibits [3H]QNB binding. Preliminary studies of furosemide effects on [3H]QNB binding to rat exorbital lacrimal gland membranes showed a similar inhibition, although the diuretic had no effect on antagonist binding to rat brain or atrial receptors.     

Distribution of (3H) QNB binding in dog gastric smooth muscle pre- and post vagotomy

Tritiated quinuclidinyl benzilate [(³H)QNB] was used to characterize muscarinic cholinergic receptors in membrane fragments prepared from the circular smooth muscle of the dog stomach. In preliminary experiments the effect of protein, incubation time, temperature and pH on QNB binding were evaluated. Muscarinic cholinergic antagonists and agonists inhibited QNB binding in a concentration-dependent manner, but the nicotinic antagonist hexamethonium and adrenergic compounds were not effective in displacing QNB from binding sites. Scatchard plot analysis of binding data showed an asymetric receptor distribution in the stomach. The cardia bound 425fmol of QNB/mg protein with a K_d of 0.05nM, the fundus 267fmol/mg protein with a K_d of 0.09nM and the antrum 147 fmol/mg protein with a K_d of 0.14nM. In a second series of experiments, binding of QNB was measured in dogs which had been vagotomized three weeks earlier. Vagotomy had no effect on the apparent K_d but disrupted the asymetric receptor distribution seen in the normal dog such that the Bmax of the cardia fell to a value of 222fmol/mg protein.     

The beta-adrenergic receptor in human lymphocytes: subclassification by the use of a new radio-ligand, (+/-)-125 Iodocyanopindolol

(±)?¹²⁵Iodocyanopindolol (ICYP), a new radio-ligand with high affinity and specificity to β-adrenoceptors was used to identify and characterized β-adrenergic receptors in human lymphocytes. Binding of ICYP was saturable with 1.56 ± 0.2 fmol ICYP specifically bound/10⁶ cells at maximal occupancy of the sites and of high affinity (K_D=57 ± 7.1pM, N=4. In contrast to ¹²⁵Iodohydroxybenzylpindolol ICYP-binding was not affected by phentolamine (up to 10^?4M) or serotin (up to 10^?5M). Analysis of inhibition of ICYP-binding via a pseudo-Scatchard-plot (“Hofstee-plot”) by β_1-selective (practocol, metaprolol) and β_2-selective (IPS 339, zinterol) adrenergic drugs resulted in linear plots suggesting the existence of a homogeneous population of β-adrenergic receptorsin human lymphocytes. From the resulting K_D-values for practolol (16.8 μM), metoprolol (4.11 μM), zinterol (0.08 μM) and IPS 339 (0.002 μM) is concluded that the β-adrenergic receptor present in human lymphocytes is of the β₂-subtype. According to its low non-specific binding and its high specificity to β-adrenergic receptors ICYP appears to be an ideal ligand for long-term studies on the regulation of β-adrenergic receptors of human lymphocytes.     

Demonstration of human platelet β-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol

The radioiodinated pindolol analogs ¹²⁵I-labeled cyanopindolol ([¹²⁵I]CYP) and ¹²⁵I-labeled hydroxybenzylpindolol ([¹²⁵I]HBP) have been used to study binding to human platelet β-adrenergic receptors. [¹²⁵I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (K_d) of 14±3 pM and maximal binding capacity (B_max) of 18±4 fmol/mg protein. Binding of [¹²⁵I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8·10⁷ s^?1·M^?1 and 3.8·10^?4 s^?1, respectively. [¹²⁵I]HBP binds to a saturable class of platelet membrane sites with a K_d of 50±10 pM and B_max of 32±6 fmol/mg protein. [¹²⁵I]HBP also binds to a saturable class of sites on intact platelets with a K_d of 58±14 pM and B_max of 24±4 molecules per platelet. Binding of [¹²⁵I]CYP and [¹²⁵I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (?) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol ? isoproterenol > epinephrine > practolol > norepinephrine > phenylephrine. These observations indicate that [¹²⁵I]CYP and [¹²⁵I]HBP bind to platelet sites which have the pharmacological characteristics of β-adrenergic receptors but which are not typical of either the β₁ or β₂ sub-type.     

Characterization of Muscarinic Receptor Subtypes in Canine Left Ventricular Membranes

Abstract

The pharmacological characteristics of muscarinic receptor (mAChR) subtypes in canine left ventricular membranes (LVM) were determined using [³H]quinuclidinyl benzilate ([³H]QNB) and [³H] N-methyl scopolamine ([³H]NMS) as ligands. Binding of [³H]QNB and [³H]NMS was saturable with respect to the radioligand concentrations. Analysis of binding isotherms by Scatchard plot showed that [³H]QNB and [³H] NMS bound to an apparently homogeneous population of mAChRs in LVM, with K_D values of 390 ± 100 and 285 ± 34 pM and B_max values of 240 ± 20 and 133 ± 9 fmol/mg protein, (n=6), respectively. The Hill coefficients for [³H]QNB and [³H]NMS binding were 0.95 ± 0.02 and 0.99 ± 0.01, respectively. Based on the competitive inhibition of [³H] ligand binding, atropine and NMS as well as the selective M₁ antagonist PZ revealed no selectivity for these mAChRs. PZ competed with [³H]QNB or [³H]NMS for a single binding site with a K_i value of 0.23 ± 0.03 μM and 0.62 ± 0.10 μM, (n = 6), respectively, which is close to the values of M² or M³ receptors. The data indicate that the M¹ receptor subtype did not exist in canine LVM. Competition of [³H] ligand binding with selective M₂ antagonists, AF-DX 116 and methoctramine and the selective M₃ antagonists, 4-DAMP and hexahydrosiladifenidol, gave a best fit for a two-binding site model. The inhibition of carbachol-mediated phosphoinositide hydrolysis by PZ, AF-DX 116 and 4-DAMP, generated an affinity profile for this response also dissimilar to that described for the classical cardiac M₂ response. Although no other muscarinic receptor mRNA has been detected in this tissue, these data suggest the presence of a second population of muscarinic sites, which may signify an M₂ receptor diversity.     

α1-Adrenergic Receptors of A Smooth Muscle Cell Line: Guanine Nucleotides Do Not Regulate Agonist Affinities

Abstract

Using [³H]-dihydroergocryptine, we have identified in membranes prepared from the DDT₁ MF-2 smooth muscle cell line a binding site with characteristics of the α₁-adrenergic receptor. Specific binding (90–95% of total binding) was saturable with a binding site concentration of 197±44 fmol/mg protein and was of high affinity with a dissociation constant of 1.7±0.4 nM. The order of agonist competition for the binding site was epinephrine (K_i=2.3±0.5μM) ≥ norepinephrine (K_i=4.4±1.3μM) ? isoproterenol (K_i=195.5±27.6μM), consistent with an α-adrenergic interaction. Computer modelling of competition curves obtained with prazosin (α₁-selective) and yohimbine (α₂-selective) indicated that the DDT₁ cell αa-adrenergic receptor was predominantly (>95%) of the α₁-subtype. Guanine nucleotides, either GTP or 5'-guanylylimidodiphosphate, did not reduce the affinity of either epinephrine of phenylephrine for the [3H]-dihydroergocryptine binding site.     

Allosteric modulation of the [3H]quinuclidinyl benzylate binding to M-cholinoreceptors in rat cerebral cortex by adrenotropic agents

The allosteric effects of adrenotropic drugs and the membranotropic agent cocaine on the kinetics of [³H]quinuclidinyl benzylate ([³H]QNB) binding to muscarine cholinoceptors of synaptosomal membranes of rat cerebral cortex were studied. In control, the best results were obtained for the model that assumes the existence of two receptor pools (with high and low affinity) with calculated parameters of the activity (K _d), amount (B _max), and mono- to dimer receptors ratio (n). For the high-affinity receptors these parameters were K _d1 = 0.18 ± 0.08 nM, B _m1 = 221.2 ± 56.7 fmol/mg protein, n ₁ = 2, and for low-affinity receptors, K _d2 = 1.33 ± 0.11 nM, B _m2 = 748.7 ± 53.3 fmol/mg protein, n ₂ = 2. Allosteric modulation of the activity of specific neurotransmitter receptors can be accomplished by changing the receptor affinity and amount as well as the proportion of mono- and dimer receptors. Under control conditions, muscarine receptors exist as dimers. In the presence of α-adrenoreceptor agonist noradrenaline and β-adrenoreceptor antagonist propranolol, only one pool of the dimer muscarine receptors remains. The number of binding sites for noradrenaline and propranolol decreases approximately by 40% and 20%, respectively. The agonist of α₂-adrenoreceptors clonidine, the antagonist of α₂-adrenoreceptors yohimbine, and a membranotropic agent cocaine change the ligand binding so that only one receptor pool remains but some of the dimer receptors become monomeric (1 < n < 2). The amount of binding sites reduces by 20%, 25%, and 45% for clonidine, yohimbine, and cocaine, respectively.     

A Comparison of the Effects of Estrogen and Cimicifuga racemosa on the Lacrimal Gland and Submandibular Gland in Ovariectomized Rats

This study aims to observe the effects of estradiol and Cimicifuga racemosa on the lacrimal gland and submandibular gland of ovariectomized rats. We randomly divided 20 adult female SD rats into four groups—a sham-operated group (SHAM), ovariectomized (OVX) group, ovariectomized group treated with estradiol (OVX+ E), and ovariectomized group treated with the isopropanolic extract of Cimicifuga racemosa (OVX+ iCR). The SHAM group and OVX group used distilled water to instead the drugs. Two weeks after ovariectomy, the estradiol and iCR were administered for 4 weeks. Next, we used H&E staining and electron microscopy to observe any histological changes in the lacrimal and submandibular glands and immunohistochemical staining to observe the expressions of cleaved caspase-3 (Casp-3) and Cu-Zn SOD (superoxide dismutase). The H&E staining find that both drugs can prevent the cells of area from shrinkage in the two kinds of gland. But under the electron microscopy, estradiol and iCR have different efficacy. Estradiol is more effective at protecting mitochondria in lacrimal gland acinar cells than iCR, and iCR is more effective at suppressing endoplasmic reticulum expansion than estradiol. Both estradiol and iCR have a similar protective function on mitochondria in the submandibular gland. The protective function of the two glands may inhibit apoptosis by suppressing the expression of Casp-3. In addition, iCR increases the expression of Cu-Zn SOD in duct system of submandibular gland. The results suggest that both estradiol and iCR confer a protective effect on the lacrimal and submandibular glands of ovariectomized rats via different mechanisms.     

Glomerular thromboxane A2/prostaglandin H2 receptors: characterization and effect of adriamycin-induced nephrotic syndrome

We have characterized the thromboxane (TX) A₂/prostaglandin (PG) H₂ receptor in glomeruli isolated from the rat using the agonist radioligand [¹²⁵I]-BPO. Binding of [¹²⁵]-BOP was highly specific, stereoselective, and to a single class of high affinity binding sites (K_d = 1/16 ± 0.22 nM and B_max = 348 ± 32 fol/mg protein; n = 6). Binding of [¹²⁵I]-BOP was competed for by the agonist ONO11113 (K_d = 50.8 ± 8.0 nM; n = 4) and the antagonists SQ29548 (K_d = 15.8 ± 1.0 nM; n = 3), L657925 (K_d = 12.1 ± 2.2 nM; n = 3) and L65796 (K_d = 1642 ± 135 nM; n = 3). I-BOP also produced a TXA₂/PGH₂ receptor-mediated rise in [CA²⁺]_i in isolated glomeruli In adriamycin-induced nephrotic syndrome in the rat, the development of proteinuria is reported to be dependent on increased renal TXA₂ production. We therefore examined whether or not changes in glomerular TXA₂/PGH₂ receptors occur between control and nephrotic rats. No changes in expression of affinity of either glomerular or platelet TXA₂/PGH₂ receptors were observed. K_d and B_max values for isolated isolated glomeruli were 1.45 ± 0.24 nM and 406 ± 72 fmol/gm for controls and 1.22 ± 0.25 nM and 321 ± 62 fmol/gm for nephrotic rats (n = 6).     

Studies of corticotropin receptors on rat adipocytes

Synthetic [¹²⁵I]-Tyr²³, Phe², Nle⁴-adrenocorticotropin (ACTH)-(1–38) ([¹²⁵I]-ACTH analog) with full biological potency and near theoretical specific radioactivity (1800 ± 75 Ci/mmol) was used to investigate ACTH receptors on isolated rat adipocytes derived from 42-day-old rats. Binding to adipocytes was studied in the presence of 1% bovine serum albumin (BSA) as well as 4% BSA. The interaction of the [¹²⁵I]-ACTH analog with adipocytes was highly specific, rapid, saturable, and reversible. Scatchard analysis of the binding data obtained in medium containing 1% BSA revealed a single class of binding sites with an apparent K_D = 170 ± 11.9 pM. Competition experiments with unlabeled ACTH also yielded a comparable value for the apparent K_D (143 ± 16.5 pm). The number of receptors per adipocyte was quite low (521–841/cell). The stimulation of lipolysis by ACTH was closely correlated with the binding, the apparent K_m being 145–177 pm. At a concentration of 4% BSA in the incubation medium, the binding curve was shifted significantly to the right (apparent K_D = 446 ± 77 pM) and the binding capacity was also significantly enhanced (1663 ± 208/cell) without any change in the apparent K_m for glycerol release (187 ± 7.1 pm).     

Solubilization of High-Affinity,Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the μ-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [³H][d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin ([³H]DAMGO), a μ-selective opioid agonist, with high affinity (K_D = 1.90 ± 0.93 nM; B_max = 629 ± 162 fmol/mg of protein). Of the membrane-associated [³H]-DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [³H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate). The solubilized receptor also bound [³H]diprenorphine, a nonselective opioid antagonist, with high affinity (K_D = 1.4 ± 0.39 nM, B_max = 920 ± 154 fmol/mg of protein). Guanosine 5′-O-(3-thiotriphosphate) did not diminish [³H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [³H]diprenorphine for the solubilized binding sites; in contrast, [d -Pen²,d -Pen⁵]-enkephalin, a δ-selective opioid agonist, and U50488H, a κ-selective opioid agonist, failed to compete with [³H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [³H]diprenorphine binding sites better fit a two-site than a one-site model with K_Dhigh = 2.17 ± 1.5 nM, B_max = 648 ± 110 fmol/mg of protein and K_Dlow = 468 ± 63 nM, B_max = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with K_D = 815 ± 33 nM, B_max = 965 ± 124 fmol/mg of protein.     

Muscarinic cholinergic binding in human erythrocyte membranes

Human erythrocyte ghosts contain a small population of muscarinic cholinergic receptors, as evidenced by their high affinity binding of radiolabeled quinuclinidinyl benzilate ([³H]QNB). The apparent K_D is 1.3 × 10^?9 M and the receptor sites are saturated at a QNB concentration of 5 nM. The number of sites is 23 fmoles/mg membrane protein. The pharmacological profile of the specific binding is similar to that of neural membranes. The binding is not stereoselective for the d and 1 isomers of QNB, a situation which prevails in the muscarinic receptors of another peripheral cholinergic system, the rat iris, but not in the central nervous system.     

Maturation of mammalian myocardial muscarinic cholinergic receptors

Biochemical alterations of the cardiac muscarinic binding sites have not been correlated with physiological observations. The development of responsiveness to acetylcholine in the fetal mouse heart occurs during the third trimester. We tested the hypothesis that the altered physiological response was related to changes in the muscarinic cholinergic binding site assayed by using the potent antagonist [³H]-quinuclidinyl benzilate (QNB). Analysis of saturation isotherms of specific (atropine displaceable) [³H]-QNB binding gave an apparent dissociation constant (K_Dapp) of 27 ± 3 (SEM) pM for adult heart. Receptor density increased significantly during the third trimester of pregnancy to 48% of adult, while the K_Dapp did not change. The increase in receptor density parallels the demonstrated increased responsiveness to acetylcholine.     

High affinity beta-2-adrenergic receptors in mononuclear leucocytes: similar density in young and old normal subjects

In normal subjects beta-adrenergic responsiveness in the cardiovascular system has been shown to be impaired with increasing age. In order to correlate reduced hormonal responsiveness to an age-related defect at the receptor level, high affinity beta-adrenergic receptors in homogenates of human mononuclear leucocytes have been studied with a (?)-³H-dihydroalprenolol (³H-DHA) binding assay. The binding sites have been characterized by rapid kinetics, saturability, structural and sterospecificity. Binding equilibrium was obtained within 16 minutes at 37° and was reversed by 50% within 10.6 minutes. In 22 healthy subjects a binding capacity of 60 ± 8 fmol/mg protein and an equilibrium dissociation constant (K_D) of 0.6 ± 0.05 nM was found. Beta-adrenergic agonists displaced ³H-DHA binding with a potency order of isoproterenol > adrenaline > noradrenaline. The (?) isomers of beta-adrenergic agonists and antagonists were one to two orders of magnitude more potent as inhibitors of ³H-DHA binding than their corresponding (+) isomers. The binding capacity and affinity of the beta-adrenergic receptors did not differ in old, as compared to young normal subjects. Leucocytes from 14 individuals 18–40 years old had an average density of 53 ± 4 fmol/mg protein, while the average density in leucocytes from 8 individuals aged 53–65 years was 67 ± 8 fmol/mg protein. The K_D was 0.6 ± 0.05 nM in both groups. In conclusion, an age-related decrease of beta-adrenergic receptor-mediated cardiovascular functions does not seem to be reflected in the properties of beta-adrenergic receptors of mononuclear leucocytes.     

Serotoninergic agonists and antagonists are the modulators of α1-Adrenoceptor activity in rat brain cortical membranes

The effects of activation and inhibition of serotonin receptors by serotonin (5-HT) and mianserin on the specific nonselective α₁-antagonist [³H]prazosine binding in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction of α₁-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and the binding of two ligand molecules to the receptor. The parameters of [³H]prazosine binding to α₁-adrenoceptors were as follows: K _d =1.85 ± 0.16 nM, B _max = 31.1 ± 0.3 fmol/mg protein, n = 2. In case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K _d1 = 0.61 ± 0.04, K _d2 = 3.82 ± 0.15 nM, B _m1 = 6.6 ± 0.7, B _m2 = 25.6 ± 0.4 fmol/mg protein, n = 2. The sensitivity of the high-affinity pool increased threefold and the sensitivity of the low-affinity pool decreased twofold as compared to the control. The value of maximal reaction (B _max) did not change. In the case of inhibition of 5HT-receptors by mianserin, radioactive ligand is bound to α₁-adrenoceptors according to the same model as in the control conditions. The affinity of α₁-adrenoceptors to [³H]prazosine decreases twofold and the concentration increases (K _d = 3.97 ± 0.12 nM, B _max = 40.0 ± 0.5 fmol/mg protein). The data suggest that α₁-adrenoceptors in rat cerebral cortex exist as a dimer. The modulatory effects of serotonin and mianserin on the specific binding of [³H]prazosine to α₁-adrenoceptors was detected, manifesting itself as changes in the binding parameters and in the general character of ligand-receptor interactions.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K _d = 1.85 ± 0.16 nM, B _max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.57 ± 0.27 nM, B _max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K _d = 1.36 ± 0.03 nM) and 84% (B _max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K _d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K _d1 = 1.13 ± 0.09, K _d2 = 6.07 ± 1.06 nM, B _m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K _d1 = 0.61 ± 0.02, K _d2 = 3.41 ± 0.13 nM, B _m1 = 1.88 ± 0.028, B _m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B _max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.     

Serotoninergic agonist and antagonist are modulators of the α2-adrenoceptor activity in rat brain cortex membranes

The influence of activation and inhibition of serotonin receptors by serotonin (5HT) and miancerin on binding of specific nonselective α₂-antagonist [³H]RX821002 in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction for α₂-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of the [³H]RX821002 binding to α₂-adrenoceptors were as follows: K _d = 1.57 ± 0.276 nM, B _max = 7.24 ± 1.63 fmol/mg protein, n = 2. In the case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K _d1 = 0.82 ± 0.06; K _d2 = 2.65 ± 0.22 nM; B _m1 = 1.65 ± 0.23; B _m2 = 4.20 ± 0.11 fmol/mg protein; n = 2. The affinity of high-affinity receptors increased twofold and the affininty of low-affinity receptors decreased by 69% as compared to control values. The concentration of high-affinity receptors decreased 4.4-fold, and of low-affinity, 1.7-fold. The value of maximal reaction (B _max) decreased by 20%. In the case of miancerin-induced inhibition of 5HT-receptors the character of ligand binding also changed; two pools of receptors were detected with the following parameters: K _d1 = 0.48 ± 0.09; K _d2 = 3.79 ± 0.71 nM; B ₁ = 0.63 ± 0.17; B ₂ = 4.75 ± 0.21 fmol/mg protein; n = 2. The affinity of high-affinity receptors pool increased by 70% and that of low-affinity receptors decreased by 76% as compared to control values. The concentration of active high-affinity and low-affinity α₂-adrenoceptors decreased by 70% and 141%, respectively. The total amount of the receptors (B _max) decreased by 26%. The data suggest that α₂-adrenoceptors in rat cerebral cortex exist as dimers. Modulatory effects of serotonin and miancerin on specific antagonist binding to α₂-adrenoceptors may be accomplished by altering the character and binding parameters of the nonselective α₂-antagonist [³H]RX821002.     

Maturation of muscarinic agonist receptors in rat developing pancreas and its relation to maximal enzyme secretion

The true K_Ds of [³H] (?) QNB binding to muscarinic receptors were found to be 4.13 and 6.43 × 10^?11 M in pancreas of 21 day fetal and adult rats. The competition curves of specific [³H] (?) QNB bound by two antagonists have shown that the affinity of these drugs did not change with age with Hill coefficients near unity. However, with the agonist carbamylcholine as the competitive drug, a more flat curve was obtained with a Hill coefficient below unity. At least two populations of carbamylcholine binding sites were found with different K_Ds: a K_H around 7 × 10^?7 M and a K_L around 3 × 10^?5 M. These two populations were present during all the developmental periods studied. The ED₅₀ of bethanechol stimulated amylase secretion did not change within the age limit studied (from 11 to 365 days). The high affinity sites for carbamylcholine would seem to be the receptor implicated in the physiological response of the pancreas.