Effect of piracetam administration on 3H-N-methylscopolamine binding in cerebral cortex of young and old rats.

Piracetam, a nootropic drug, has been used for some time in Alzheimer's disease for its facilitatory effect on learning and memory. Rats treated with piracetam (500 mg/kg, p.o.) daily, during 1 and 2 weeks, showed a significant increase in muscarinic receptor number (Bmax) and in the dissociation constant values (Kd) in the cerebral motor cortex, in binding studies using 3H-NMS as ligand. The effect was observed not only in young rats (control- Bmax = 663.4 fmol/mg protein, Kd = 0.45 nM; treated- Bmax = 961.9 fmol/mg protein, Kd = 0.82 nM) but also in aged animals (control- Bmax = 628.0 fmol/mg protein, Kd = 0.47 nM; treated-Bmax = 747.6 fmol/mg protein, Kd = 0.84 nM). Since piracetam does not interact with muscarinic receptors, the reason for its effect expressed as the enhanced number of brain muscarinic receptors is not clear but could be the result of stimulation of phospholipid synthesis and thus would represent an indirect action of the drug.     

Modulation of A2A adenosine receptor(s) by K+(ATP) channels in bovine brain striatal membranes

The modulation of adenosine receptor with K+(ATP) channel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10(-4) M. In the presence of 10(-5) M glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10(-4) M. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd = 10.6+/-1.71 nM; Bmax = 221.4+/-6.43 fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10(-5) M glibenclamide; (Kd = 1.3+/-0.22 nM; Bmax = 74.3+/-2.14 fmol/mg protein; and Kd = 8.9+/-0.64 nM; Bmax = 243.2+/-5.71 fmol/mg protein), indicating modulation of adenosine A2A receptors by glibenclamide. These studies suggest that the K+(ATP) channel blocker, glibenclamide, modulated the adenosine A2A receptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that the interactions between K+(ATP) channels and adenosine receptors.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

A muscarinic receptor type in human lymphocytes: a comparison of 3H-QNB binding to intact lymphocytes and lysed lymphocyte membranes

Human blood lymphocytes from normal blood donors exhibited specific binding of the muscarinic antagonist 3H-quinuclidinyl benzilate (3H-QNB). The 3H-QNB binding to intact viable lymphocytes as well as to lysed lymphocyte membranes "P2" was saturable and displaceable by both muscarinic agonists and antagonists. For the lysed lymphocyte membranes "P2" a single binding site with a Bmax of 109 pmol/g protein and a Kd of 15 nM was obtained. Intact viable lymphocytes also showed one binding site with a Kd of 24 nM and a Bmax of 1556 pmol/g protein. The higher Bmax value might be explained in terms of uptake of the ligand when using intact cells or through loss of binding sites when using lysed lymphocyte membranes "P2". IC50 values were lower by a factor of 10(2) for atropine and scopolamine and by 10(4) for pirenzepine when lysed lymphocyte membranes "P2" were used instead of intact viable lymphocytes.     

Iminodipropionitrile-Induced Dyskinesia in Mice: Striatal Calcium Channel Changes and Sensitivity to Calcium Channel Antagonists

Administration of 3,3'-iminodipropionitrile (IDPN) (1 g/kg, i.p. for 3 days) in mice leads to the development of a characteristic syndrome consisting of lateral and vertical head and neck movements, hyperactivity, random circling, increased locomotor activity, and increased startle response. Nifedipine, verapamil, and diltiazem (10 mg/kg) inhibited significantly the symptoms of IDPN-induced dyskinesia. However, there was no change in the affinity (KD) or the density of PN 200-110 binding sites (Bmax) in whole brains of IDPN-treated mice. Similarly, the K(+)-depolarization-dependent Ca2+ uptake in synaptosomes from whole brain, cortex, or striatum was not altered following IDPN treatment. However, IDPN caused a significant increase in the Bmax value (from 157 +/- 7 fmol/mg to 237 +/- 31 fmol/mg in control and treated groups, respectively) of PN 200-110 binding to the striatum without change of KD value (38 +/- 4.7 pM versus 33 +/- 1.6 pM). IDPN also caused a slight but significant decrease in the KD value (from 68 +/- 10.1 pM to 45 +/- 4.5 pM in control and treated groups, respectively), without significant change of Bmax value (563 +/- 51 fmol/mg versus 485 +/- 41 fmol/mg) of PN 200-110 binding to the cortex. IDPN did not alter omega-conotoxin binding in whole brain, striatum, or cortex. The behavioral effects of chronic IDPN treatment as inhibited by L-type calcium channel antagonists and this may be associated with the observed increase in striatal L-type calcium channels.     

Reserpine-induced post-receptor reduction in muscarinic-mediated airway smooth muscle contraction

Radioligand binding was conducted on airways of the rat and human, surgically subdivided into trachea, lung airways, and parenchyma. 3H-QNB bound uniformly to receptors in separate sections of the rat and human airway. Receptor densities generally were ranked: lung airways greater than trachea greater than parenchyma. Receptor subtypes were identified mostly by pirenzepine displacement of bound 3H-QNB. The rat trachea, and rat and human lung airways had a uniformly low affinity for pirenzepine while rat and human parenchyma demonstrated both high and low affinity pirenzepine binding. Inhibition of methacholine-stimulated smooth muscle contraction by the M1 receptor antagonist, pirenzepine, and M2 receptor antagonist, gallamine, was studied in rat trachea and bronchus in vitro. Schild plot pA2 values were compatible with low potency antagonism, thereby favoring the presence of M3 receptors at these smooth muscle sites. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) produced a decrease in peak tension in response to methacholine without changing the muscarinic receptor character (Kd 3H-QNB), population density (Bmax in fmol mg-1 protein), or function (methacholine EC50). These results indicate that muscarinic receptor heterogeneity exists in the airway of both laboratory rat and man. While the muscarinic receptor subserving airway smooth muscle contraction appears to be the M3 subtype, decreased contractile responses to methacholine by trachea and bronchus from reserpine-treated rats were receptor independent.     

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.     

Partial functional reconstitution of the cardiac muscarinic cholinergic receptor

Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system. Specific [3H](-)-quinuclidinyl benzilate binding to the reconstituted receptor was found to be saturable with a Kd (dissociation constant) equal to 48 +/- 4 pM and a Bmax (maximal density of binding sites) equal to 50 +/- 5 fmol/mg of protein. Competitive binding studies indicated that the reconstituted receptors showed stereoselectivity and drug specificity consistent with a high-affinity muscarinic receptor. Agonist binding to the reconstituted receptor was decreased by the addition of guanyl-5'-yl imidodiphosphate. Sixty per cent of the reconstituted receptors were found to be integral membrane proteins. The molecular weight of the reconstituted receptor as determined by sodium dodecyl sulfate-gel electrophoresis was 76,000 +/- 2,000 and was identical to the molecular weight of the muscarinic receptor in the original cardiac membranes. The data indicate that a partially functional, intact muscarinic receptor was reconstituted into human erythrocyte acceptor membranes and that membrane constituents may be required to stabilize the receptor in a high-affinity state for antagonists.     

Calcium channel binding in nerves and muscle of canine small intestine

Using [3H]-nitrendipine (Nit) and [125I]-omega conotoxin (w-CTX), the cellular and subcellular distribution of calcium channel subtypes in the homogenates of canine small intestinal circular muscle was studied. Nit. bound to the membranes from the circular smooth muscle cells (PM) and to the synaptosomal membranes from the deep muscular plexus (DMP); the Kd and Bmax values of Nit binding from these two sources were similar (Kd 0.4 +/- 0.16 nM and 0.77 +/- 0.24 nM; Bmax 206 +/- 22 and 192 +/- 39 fmol/mg of protein in DMP and PM respectively). w-CTX, however, bound only to the DMP (Kd 18.41 +/- 7.5 pM, Bmax 265 +/- 36 fmol/mg of protein). In DMP, nifedipine (10(-6) M) failed to interact with the binding of w-CTX; similarly, no modulation of Nit binding with unlabelled w-CTX (10(-7) M) could be detected. Therefore w-CTX and Nit binding sites represent two distinct, non-interactive and differentially distributed binding sites in canine small intestine.     

Identification of B2 bradykinin binding sites in the guinea pig nasal turbinate

Specific high affinity BK binding sites in the nasal turbinate of the guinea pig have been demonstrated. Specific [3H]BK binding (10-330 pM) was saturable, and nonlinear least squares analysis indicated the presence of a high affinity binding site with a Kd value of 60 (50-78) pM and a Bmax value of 13.1 = 2.0 fmol/mg protein. In inhibition experiments, D-Phe7-BK (a B2 antagonist) inhibited [3H]BK binding with a Ki value of 23 nM, while des-Arg9[Leu8]-BK (a B1 antagonist) had no effect up to a concentration of 10 microM. These studies indicate the presence of B2 BK receptors in the guinea pig nasal turbinate.     

Selective inhibition of the binding of 3H-(3-MeHis2) thyrotropin releasing hormone to rat amygdala membranes by some naturally occurring cannabinoids

The effect of naturally occurring cannabinoids, delta 9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD), on the brain receptors for thyrotropin releasing hormone (TRH) was investigated. TRH receptors were labeled with 3H-(3-MeHis2)TRH (3H-MeTRH). 3H-MeTRH bound specifically to rat brain membranes at a single high affinity site with a Bmax value of 49.2 +/- 0.96 fmol per mg protein and a Kd value of 3.83 +/- 0.12 nM. The binding of 3H-MeTRH to whole brain membranes was inhibited when rats were injected intraperitoneally with 3 to 30 mg/kg of THC. The extent of inhibition in the binding at 10 and 30 mg/kg was similar. THC (10 mg/kg) significantly inhibited the binding of 3H-MeTRH to amygdala membranes but did not affect the binding to membranes prepared from hippocampus, septum, cortex, striatum and the rest of the brain. THC, CBN and CBD in doses of 3 to 30 mg/kg did not affect the binding of 3H-MeTRH to hypothalamic membranes. All the three cannabinoids at 30 mg/kg inhibited the binding of 3H-MeTRH to amygdala membranes. The inhibition in the binding of 3H-MeTRH by the cannabinoids was due to changes in the Kd values but the Bmax values remained unchanged. It is concluded that both psychotomimetic and nonpsychotomimetic cannabinoids inhibit the binding of 3H-MeTR to amygdala membranes selectively, which is accomplished by decreases in the affinity of the ligand to receptors, and the amygdala may be an important brain area in some of the actions of cannabinoids.     

Amplification of the rat M2 muscarinic receptor gene by the polymerase chain reaction: functional expression of the M2 muscarinic receptor

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH beta APr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H] (-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411 +/- 50 fmol/10(6) cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H] (-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 microM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.     

Identification and characterization of melatonin binding sites in the gastrointestinal tract of ducks.

Utilizing 2-[125I]iodomelatonin as the radioligand, melatonin binding sites were identified and characterized in the jejunum of ducks. These sites were found to be reversible, saturable, specific and exhibited high affinity for melatonin. Scatchard analyses have established the equilibrium dissociation constant (Kd) for tissues collected during mid-photophase to be 40.9 +/- 7 pM and the maximum quantity of binding sites (Bmax) to be 2.0 +/- 0.4 fmol/mg protein while Kd of samples collected during mid-scotophase was found to be 54.1 +/- 10 pM with a corresponding Bmax of 1.5 +/- 0.3 fmol/mg protein. These Kd values are in good proximity to the kinetically derived equilibrium dissociation constant of 47.3 +/- 20 pM. No significant difference in Kd or Bmax was detected between the mid-light and mid-dark samples. Pharmacological profile of these binding sites, developed by their interactions with other indoles and compounds, indicated that these binding sites are highly specific for melatonin. Subcellularly, different densities of binding sites were localized to various fractions in the following order: nuclear greater than microsomal greater than mitochondrial greater than cytosolic. These binding sites in the jejunum might be the receptors accountable for promoting paracrine activities for the locally synthesized gastrointestinal melatonin and/or responsible for eliciting hormonal actions via interactions with melatonin of pineal origin.     

Increase in muscarinic acetylcholine receptors in mouse intestine by hexamethonium treatment

The effects of hexamethonium (C6) administration on muscarinic acetylcholine receptors (mACh-R) in the intestine and brain of mice were investigated. Mice were treated with C6 with an osmotic mini-pump (330 mg/kg/day) for one week and then the binding of 3H-quinuclidinylbenzilate (3H-QNB) in the intestine and brain were assayed. This treatment increased the maximum specific binding (Bmax) of 3H-QNB from 160 to 320 fmoles/mg protein in the ileum and from 190 to 340 fmoles/mg protein in the rectum, without affecting the KD values in these regions. On the contrary, C6 treatment did not change the Bmax or KD value in brain tissues. This C6 treatment increased the sensitivity of the contractile response of the intestine to muscarinic agonists, possibly by increasing mACh-R.     

Identification and characterization of atrial natriuretic factor receptors in the rat retina

The characteristics of atrial natriuretic factor (ANF) receptors where studied in rat retinal particulate preparations. Specific 125I-ANF binding to retinal particulate preparations was greater than 90% of total binding and saturable at a density (Bmax) of 40 +/- 8 fmol/mg protein with an apparent dissociation constant (Kd) of 6.0 +/- 2.0 pM (n = 3). Apparent equilibrium conditions were established within 30 min. The Kd value of 125I-ANF binding calculated by kinetic analysis was 4.0 pM. The Bmax of 60 +/- 10 fmol/mg protein and the Kd of 5 +/- 2 pM, calculated by competition analysis, were in close agreement with the values obtained from Scatchard plots or kinetic analysis. The 125I-ANF binding to retinal particulate preparations was not inhibited by 1 microM concentration of somatostatin, vasopressin, vasoactive intestinal peptide, adrenocorticotropin, thyrotropin releasing hormone, or leu-enkephalin. The rank order of potency of the unlabelled atrial natriuretic peptides for competing with specific 125I-ANF (101-126) binding sites was rANF (92-126) greater than rANF (101-126) greater than rANF (99-126) greater than rANF (103-126) greater than Tyro-Atriopeptin I greater than hANF (105-126) greater than rANF (1-126). Similar results have been obtained in peripheral tissues and mammalian brain, indicating that central and peripheral ANF-binding sites have somewhat similar structural requirements. Affinity cross-linking of 125I-ANF to retinal particulate preparations resulted in the labelling of two sites of molecular weight 140 and 66 kDa, respectively. This demonstration of specific high-affinity ANF receptors suggests that the peptide may act as a neurotransmitter or neuromodulator in the retina.     

Modification of morphine-induced change in striatal (3H)-spiroperidol binding and stereotype behavior by cyclo(Leu-Gly)

Recent reports from our laboratories have indicated that the peptide cyclo(Leu-Gly), an analog of MIF (Pro-Leu-Gly-NH₂), administered prior to chronic exposure to morphine, prevents the development of both analgesic tolerance and some signs of physical dependence. The same peptide treatment also prevented the development of morphine-induced increases in certain behavioral responses to the dopamine agonist apomorphine. The present study investigated behavioral (stereotypy) and neurochemical receptor changes (specific (³H)-spiroperidol binding) occuring in the rat striatal dopamine (DA) system following chronic morphine treatment with and without prior cyclo(Leu-Gly)administration. While chronic morphine treatment (s.c. 5 pellet implant for 3 days, each pellet contained 65 mg morphine free base) did not alter the total number of high-affinity striatal (³H)-spiroperidol binding sites (28 fmol/mg tissue), it did increase the affinity of the receptor for the ligand (K_D decreased from 40 to 24 pM). Cyclo(Leu-Gly) (8 mg/kg) prevented the morphine induced increase in dopamine receptor affinity. In parallel, cyclo(Leu-Gly) prevented the increase in apomorphine-induced stereotypy which was observed in chronic morphine treated rats. The peptide alone did not alter any of the binding characteristics. These data suggest that the ability of the peptide to block the development of physical dependence induced by morphine may involve the ability of the peptide to interfere with morphine-induced changes in dopaminergic systems.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

Effect of reduced oxygen intermediates on sarcolemmal muscarinic receptors from canine heart

The effect of oxygen free radicals generated by xanthine-xanthine oxidase system and hydrogen peroxide were investigated on cardiac muscarinic cholinergic receptors. We have used highly enriched sarcolemmal preparations isolated from canine myocardium. Exposure of the sarcolemma to oxygen free radicals by xanthine-xanthine oxidase system resulted in a significant (P less than 0.05) decrease of Bmax of (3H)-QNB (4.66 +/- 0.51 to 2.68 +/- 0.22 pmoles/mg protein). Addition of superoxide dismutase (SOD) and catalase (10 micrograms/ml) resulted in a significant reversal of Bmax value to 3.72 +/- 0.39 pmoles per mg protein (p less than 0.05). However, the affinity constants of dissociation (KD) were not altered appreciably with the exposure to oxygen free radicals with or without scavengers. Hydrogen peroxide significantly depressed 3H-QNB binding to the receptors in a dose-dependent manner in a concentration range between 4.41 mM -441 mM. This depression was completely inhibited by 10 micrograms/ml catalase. The study demonstrates that the oxygen free radical species are capable of disrupting (3H)-QNB binding to the cardiac muscarinic receptors.     

beta-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.