The binding of 3H-acetylcholine to cholinergic receptors in bovine cerebral arteries

Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, 3H-acetylcholine (ACh), as the ligand. Specific binding of 3H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (KD) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of 3H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC50 values of cholinergic drugs for 3H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC50 values of nicotinic cholinergic agents such as nicotine, cytisine and alpha-bungarotoxin exceeded 50 microM. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic receptors in these arteries.     

Identification of α2-Adrenergic Receptors in Chicken Pineal Gland Using [3H]Rauwolscine

The norepinephrine-induced inhibition of avian pineal N-acetyltransferase activity appears to be mediated by alpha 2-adrenergic receptors. In this study, alpha 2-adrenergic receptors in the chicken pineal gland were directly identified by radioligand binding. Membrane preparations of pineal glands from chickens from 1 to 6 weeks of age were examined using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, to characterize the binding sites. The results indicate no ontological change in either the affinity (KD) or density of receptor binding sites (Bmax) during the time span examined. The binding was saturable and of high affinity with a mean KD of 0.27 +/- 0.01 nM and a mean Bmax of 242 +/- 12 fmol/mg protein. Further characterization of these binding sites indicated that the alpha 2-adrenergic receptor is of the alpha 2A subtype, since prazosin and ARC-239 bound with low affinities and oxymetazoline bound with high affinity.     

Inhibitory actions of muscarinic cholinergic receptor agonists on serotonin N-acetyltransferase in bovine pineal explants in culture

We have previously identified muscarinic cholinergic receptors in the bovine pineal gland with a K_D value of 0.423±0.01 nM and a B_max value of 69.75±20.91 fmol/mg protein. Similarly, we have shown that the bovine pineal gland possesses a specific choline acetyltransferase with an activity of 0.034±0.004 nmol/mg protein/min. In order to delineate the function of these cholinergic receptor sites, we have studied the effects of muscarinic cholinergic receptor agonists on the activity of serotonin N-acetyltransferase, the melatonin synthesizing enzyme. Cholinergic receptor agonists such as methacholine (10 M), carbachol (10 M), and oxotremorine (10 M) inhibited the activity of serotonin N-acetyltransferase in the bovine pineal explants in culture, from a control value of 5.02±0.45 to 1.25±0.25, 1.30±0.15, and 1.22±0.20 pmol/mg protein/min, respectively. These inhibitory effects were blocked by muscarinic cholinergic receptor antagonists such as atropine (20 M) or QNB (20 M). The presence of high affinity muscarinic cholinergic binding sites, of a specific choline acetyltransferase, along with an inhibitory action of cholinomimetic agents on the activity of serotonin N-acetyltransferase, are interpreted to suggest that muscarinic cholinergic fibers may modulate the synthesis and actions of pineal melatonin.     

Comparison of muscarinic and beta adrenergic receptors between bovine peripheral lung and tracheal smooth muscles: a striking difference in the receptor concentration

This study was undertaken to compare the activity of muscarinic and beta adrenergic receptors in bovine peripheral lung to the corresponding receptor activity in tracheal smooth muscle. We used [3H] quinuclidinyl benzilate (QNB) and [3H]dihydroalprenolol (DHA) to measure muscarinic and beta receptor activity, respectively. Binding to QNB and DHA at 25 degrees C was rapid, reversible, saturable and of high affinity. The order of potency for cholinergic and adrenergic agents competing for binding was compatible with muscarinic and beta 2 adrenergic potencies. We found that the concentration of muscarinic receptor binding sites was 37-fold greater in the tracheal muscle preparation (2805 +/- 309 fmol/mg protein) than in the peripheral lung preparation (76 +/- 28 fmol/mg protein). Unlike muscarinic receptors, the lung contained 8-fold higher concentration of the beta adrenergic receptors than did the tracheal muscle (1588 +/- 417 vs. 199 +/- 42 fmol/mg protein). The dissociation constant or the agonist's inhibitory constant (Ki) for either receptor binding site, however, was not significantly different between the two tissues. Furthermore, in vitro contraction studies showed that the response of tracheal muscle strips to methacholine was markedly greater than the response of peripheral lung strips, a finding consistent with the QNB binding result. The muscle but not the peripheral lung strip exhibited a relaxing response to epinephrine. Our data indicate a striking quantitative difference in muscarinic and beta adrenergic receptors between lung tissue and tracheal muscle, and that each receptor in the lung is qualitatively similar to the corresponding receptor in the muscle.     

Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors

The present study shows that N-[3H]methylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD = 5.0 nM; Bmax = 8.2 fmol/mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high-affinity binding sites (KD = 4.8 nM; Bmax = 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]pirenzepine. In contrast, the M2 ligand, [3H]AF-DX 116, appears to bind to two classes of binding sites, i.e., a high-affinity (KD = 3 nM)/low-capacity (Bmax = 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD = 28.4 nM) and higher capacity (Bmax = 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4-diphenylacetoxy-N-methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD = 0.5 nM)/low capacity (Bmax = 19.5 fmol/mg of protein) and one of low affinity (KD = 57.5 nM)/high capacity (Bmax = 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration-dependent manner, and the maximal increase in acetylcholine release (59.0-68.0%) occurred at 10(-7) M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and tetrodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-beta-erythroidine, d-tubocurarine, and kappa-bungarotoxin, but was insensitive to either atropine or alpha-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic Cholinergic Receptors in Human Foetal Brain: Characterization and Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Frontal Cortex

Twenty-two frontal cortices from normal human foetal brains of gestational ages ranging from 16 to 40 weeks and five postnatal brains ranging from 5 to 50 years were analysed for the ontogeny of muscarinic receptors using [3H]quinuclidinyl benzilate (QNB) as the ligand. QNB binding sites were shown to be stable up to 4 1/2 months of storage at -70 degrees C. QNB binding was characterized in frontal cortices of 28-week-old foetal brains as muscarinic receptors by the following criteria: (1) it was localised mainly in particulate fraction; (2) binding was saturable at a concentration of 1.5 nM; (3) the cholinergic antagonists atropine and scopolamine competed for the binding, with IC50 values of 1 and 0.8 nM, respectively. The agonists oxotremorine, carbachol, and pilocarpine gave IC50 values of 1, 15 and 18 microM, respectively. Nicotinic receptor ligands and noncholinergic drugs could not compete for the binding. Bimolecular association and dissociation rate constants for the reversible binding are 6.23 X 10(8) M-1 X min-1 and 2.0 X 10(-2) X min-1, respectively. The equilibrium dissociation constant is 33 pM. The KD obtained by saturation binding data is 103 pM. Ontogeny of muscarinic receptors showed three distinct phases: In phase I, they appear between 16 and 18 weeks [average concentration 109 fmol/mg protein of total particulate fraction (TPF)] and slowly increase up to 20 weeks (average concentration 147 fmol/mg protein TPF). Phase II is a lag period between 20 and 24 weeks at which time receptor concentration does not change perceptibly (average concentration (67 fmol/mg protein TPF).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.     

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.     

The Intact Human Neuroblastoma Cell (SH-SY5Y) Exhibits High-Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols

The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.     

Solubilization of human platelet vasopressin receptors

The human platelet membrane receptor for vasopressin (AVP) has been solubilized with the cholic acid derivative detergent 3-( [3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. Rapid and simple separation of free tritiated AVP ( [3H]AVP) from the solubilized receptor-hormone complex was done by filtration through polyethylenimine-treated filters. [3H]AVP binds to this soluble receptor with an equilibrium dissociation constant of 11.03 +/- 1.86 nM and a maximal number of binding sites = 288 +/- 66 fmol/mg protein while the corresponding values of the membrane-bound receptor are 1.62 +/- 0.21 nM and 237 +/- 38 fmol/mg of protein, respectively. The Ki value for native AVP derived from competition experiments is 11.02 +/- 2.05 nM for the soluble receptor. Competition experiments with specific vascular and renal antagonists confirm that the solubilized receptor belongs to the V1-vascular subtype.     

Effects of chronic nicotine treatment on nicotinic receptors in the rabbit urinary bladder

Nicotine induced a phasic contraction in the rabbit urinary bladder. The response was abolished by hexamethonium and partially reduced by atropine and capsaicin. Simultaneous atropine and capsaicin treatment did not abolish the contraction. These findings suggest that the response to nicotine is due to acetylcholine, tachykinins, and unknown mediator release. In contrast, nicotine-induced contraction diminished following the chronic nicotine treatment without a change of its pharmacological properties. These results suggest the possibility that chronic nicotine treatment causes a decrease in nicotinic receptor numbers. Therefore, the binding properties of (-)-[3H]nicotine on rabbit urinary detrusor muscle membrane fractions were studied to evaluate the effects of chronic nicotine treatment on nicotinic receptors. Specific (-)-[3H]nicotine binding reached saturation and Scatchard plots were curvilinear, suggesting the existence of two different affinity sites for (-)-[3H]nicotine. Dissociation constants (KD) and maximum binding sites (Bmax) were KD1 = 4.91 +/- 1.88 nM, Bmax1 = 2.42 +/- 0.22 fmol/mg protein and KD2 = 263 +/- 56 nM, Bmax2 = 25.0 +/- 4.3 fmol/mg protein. In urinary bladder membrane fractions from chronic nicotine-treated rabbits, KD and Bmax values were KD1 = 3.96 +/- 0.38 nM, Bmax1 = 1.07 +/- 0.25 fmol/mg protein and KD2 = 249 +/- 12 nM, Bmax2 = 10.8 +/- 1.5 fmol/mg protein. Dissociation constants for both sites following chronic nicotine treatment did not change but maximum binding site numbers for both sites significantly decreased (p less than 0.05). These results suggest that the decrease in contractile response evoked by nicotine after chronic nicotine treatment in rabbit urinary bladder is due to a decrease in numbers of nicotinic receptors.     

Neurotensin receptors on the rat liver plasma membranes

Neurotensin (NT) is now classified as a brain-gut peptide in the central nervous system and gastrointestinal tract. In the present study, we characterized the NT receptors on the rat liver plasma membranes. The specific binding of [3H]NT was time dependent, reversible, and saturable. Scatchard analysis of the specific binding data yielded two classes of binding sites, a high affinity site and a low affinity site. The average maximum number of binding sites (Bmax) amounted to 13.3 +/- 1.1 fmol/mg protein at high affinity site and 122.3 +/- 21.5 fmol/mg protein at low affinity site, respectively. The dissociation constant (Kd) had values of 0.39 +/- 0.01 nM at high affinity site and 8.1 +/- 1.1 nM at low affinity site, respectively. The amount of specifically bound [3H]NT was significantly reduced in the presence of mono and divalent cations, EDTA, EGTA and a peptidase inhibitor bacitracin, NT1-13 competed with [3H]NT for its binding site with an IC50 of 0.19 nM at high affinity site (0.2 nM concentration of [3H]NT) and 0.7 nM at low affinity site (4.0 nM concentration of [3H]NT). Xenopsin, a NT analogue separated from the skin of Xenopus laevis, was equipotent (IC50 0.75 nM) with NT1-13 at 4.0 nM concentration of [3H]NT. C-terminal sequence of NT contains the structure necessary for interaction with NT binding sites whereas N-terminal sequence had no binding activity. Since NT has a hyperglysemic and a hypercholesterolemic effects in rats, these NT receptors on the rat liver plasma membranes may be involved in the hyperglycemia and/or hypercholesteroremia induced by NT.     

Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

Biochemical Characterization of the Nicotinic Cholinergic Receptors in Human Brain: Binding of (—)-[3H]Nicotine

(-)-[3H]Nicotine was found to bind specifically to membranes of human brains obtained at autopsy. The binding was stereospecific, (-)-nicotine being 40 times more potent than (+)-nicotine in displacing labeled (-)-nicotine. Saturation binding studies revealed the presence of two binding sites with dissociation constant (KD) values of 8.1 and 86 nM, and maximum binding capacity (Bmax) values of 36 and 90 fmol/mg protein, respectively. In competition studies, nicotinic agonists were 1,000 times more potent than ganglionic, neuromuscular, and muscarinic blocking drugs in displacing labeled (-)-nicotine. IC50 values for cholinergic drugs of (-)-[3H]nicotine binding were as follows: (-)-nicotine, 0.51 nM; acetylcholine, 12.6 nM; (+)-nicotine, 19.9 nM; cytisine, 27.3 nM; and carbachol, 527 nM. IC50 values of alpha-bungarotoxin, hexamethonium, d-tubocurarine, and atropine were larger than 50 microM. (-)-[3H]Nicotine binding was highest in the nucleus basalis of Meynert and thalamus and lowest in the cerebral cortex and caudate in the brain regions tested. These results suggest that nicotinic cholinergic receptors are present in human brain and that there are regional differences in the density of these receptors.     

Alterations in 5-HT(1A) receptors and adenylyl cyclase response by trazodone in regions of rat brain

The in vivo effect of trazodone on the density of [(3)H]5-HT binding sites and 5-HT(1A) receptors and adenylyl cyclase (AC) response was studied in regions of rat brain. The chronic administration of trazodone (10 mg/Kg body wt, 40 days) resulted in a significant downregulation of [(3)H]5-HT binding sites and 5-HT(1A) receptors in cortex and hippocampus. Trazodone significantly (p < 0.0001) decreased the density of [(3)H]5-HT binding sites in cortex (42.6 +/- 3.6 fmol/mg protein, 65%) and hippocampus (12.6 +/- 1.6 fmol/mg protein, 87%) when compared to control values of 121.9 +/- 5.4 and 99.3 +/- 7.5 fmol/mg protein in these regions, respectively. Similarly there was a significant (p < 0.0001) decrease in the density of 5-HT(1A) receptors in both cortex (7.2 +/- 0.5 fmol/mg protein, 70%) and hippocampus (6.3 +/- 1.2 fmol/mg protein, 79%) when compared to control values of 24.2 +/- 2.1 and 30.6 +/- 3.7 fmol/mg protein, in these regions respectively. However, the affinity of [(3)H]5-HT to 5-HT binding sites (1.83 +/- 0.26 nM, p < 0.0001) and [(3)H]8-OH-DPAT to 5-HT(1A) receptors (0.60 +/- 0.06 nM, p < 0.05) was significantly decreased only in cortex when compared to the control K(d) values of 0.88 +/- 0.04 nM and 0.47 +/- 0.02 nM in these regions, respectively.The basal AC activity did not alter in treated rats, where as, the inhibition of forskolin-stimulated AC activity by 5-HT (10 microM) was significantly (p < 0.0001) decreased both in cortex (43%) and hippocampus (40%) when compared to control levels. In conclusion, chronic treatment with trazodone results in downregulation of 5-HT(1A) receptors in cortex and hippocampus along with concomitant increased AC response, suggesting the involvement of 5-HT(1A) receptor-mediated AC response in the mechanism of action of trazodone.     

Subcellular Localization and Characterization of Vasopressin Binding Sites in the Ventral Septal Area, Lateral Septum, and Hippocampus of the Rat Brain

[Arg8]-Vasopressin (AVP) has been shown to exert characteristic central physiological actions in the ventral septal area of the rat brain. This study reports the characterization of receptors for AVP in synaptic plasma membranes prepared from the ventral septal area, the lateral septum, and the hippocampus. Binding of [3H]AVP was temperature and time dependent, linearly related to protein concentration, saturable, and specific. Scatchard plot analysis suggested the presence of a population of binding sites in the three brain areas with dissociation constants and maximal binding capacities, respectively, of 1.06 +/- 0.39 nM and 24.0 +/- 7.01 fmol/mg of protein (mean +/- SEM; n = 3 for the ventral septal area, 0.92 +/- 0.13 nM and 47.0 +/- 4.96 fmol/mg of protein (n = 3) for the lateral septum, and 0.91 +/- 0.14 nM and 25 +/- 5.02 fmol/mg of protein (n = 3) for the hippocampus. In all three brain regions, the rank order of potencies of several vasopressin analogs, unrelated peptides, and other compounds for competitive displacement of ligand indicated a receptor with properties resembling those of the V1-like receptor for AVP. These data document the presence of a high-affinity, V1-like vasopressin receptor in the rat ventral septal area for which the pharmacological properties are similar to those previously reported in physiological studies.     

19-hydroxyandrostenedione does not modulate [3H]aldosterone binding to human mononuclear leucocytes and rat renal cytosol

To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.