Allosteric effects of atropine and carbachol on the activity of α<Subscript>2</Subscript>-adrenoceptors in rat brain cortex membranes

Activation and inhibition of muscarinic cholinoceptors by atropine and carbachol are shown to exert allosteric effects on the binding of specific nonselective α₂-adrenoceptor antagonist [³H]RX821002 in rat brain cortex membranes. The ligand-receptor interaction for α₂-adrenoceptors corresponded to the model suggesting the presence of one homogeneous pool of receptors with two specific binding sites. The parameters of the [³H]RX821002 binding were as follows: [³H]RX821002 -K _d = 1.94 ± 0.08 nM, B _max = 13.4 ± 1.8 fmol/mg protein, n = 2. The inhibition of muscarinic cholinoceptors by atropine induced an increase of affinity (K _d = 1.36 ± 0.12 nM) and a decrease of the α2-adrenoceptor density (B _max = 10.18 ± 0.48 fmol/mg protein). The muscarinic cholinoceptor agonist carbachol induced an increase of the affinity (K _d = 1.56 ± 0.05 nM) and quantity of binding sites (B _max = 16.61 ± 0.29 fmol/mg protein). As a result, under the influence of atropine and carbachol, the efficiency of binding (E = B _max/2K _d) increased from 3.50 ± 0.40 to 5.60 ± 0.79 and 6.86 ± 0.20 fmol/mg protein/nM, respectively. The data suggest that α₂-adrenoceptors exist in rat brain cortex as homodimers.     

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).     

[3H] 5,7-Dichlorokynurenic Acid,a High Affinity Ligand for the NMDA Receptor Glycine Regulatory Site

Abstract

The NMDA subtype of glutamate receptors is allosterically linked to a strychnine-insensitive glycine regulatory site. Kynurenic acid and its halogenated derivatives are non-competitive NMDA antagonists acting at the glycine site. We have prepared [³H] 5,7-dichlorokyrurenic acid (DCKA) as an antagonist radioligand and have characterized its binding. 3-Bromo-5,7-DCKA was catalytically dehalogenated in the presence of tritium gas and HPLC purified to yield [³H] 5,7-DCKA with a specific activity of 17.6 Ci/mmol. [³H] 5,7-DCKA bound to rat brain synaptosomes with a K_d of 69 ± 23 nM and B_max = 14.5 ± 3.2 pmoles/mg protein. Binding was 65–70% specific at 10 nM [³H] 5,7-DCKA. This ligand is thus more selective and has higher affinity than [³H] glycine, in addition to being an antagonist.     

Two Classes of [3H]Spiperone Binding Sites in Bovine Neurohypophysis: D-2 Receptors and Putative 5-Ht2 Receptors

Abstract

Binding of [³H]spiperone was studied in membranes obtained from bovine neurohypophyses devoid of intermediate lobe tissue. Non-linear Scatchard plot suggested the presence of more than a single class of binding sites. Competition experiments using ketanserin, a ligand selective for 5-HT₂ receptors, were carried out to ascertain whether serotonergic, in addition to dopaminergic receptors, might be responsible for the heterogeneity of [³H]spiperone binding. Computer-assisted modeling suggested the presence of two classes of binding sites for ketanserin (K_a = 1.6 ± 0.2 and 366.7 ± 20.5 nM, respectively). The K_a value for ketanserin binding to the high-affinity sites, as well as the K_a of [³H]spiperone for these sites suggested by the 2 sites model indicate that they represent serotonin 5-HT₂ receptors. The [³H]spiperone K_a at the ketanserin low-affinity sites (65 ± 7 pM) and the rank order of inhibitory potencies for several antagonists show that the lowaffinity sites represent dopamine D-2 receptors.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Identification and Characterization of Specific Binding Sites for Somatostatin in Cytosol of Bovine Cystic Duct Mucosa

Abstract

Specific binding sites for somatostatin have been detected in cytosolic fraction of bovine cystic duct mucosa. At 37°C, the interaction of ¹²⁵I-Tyr¹¹-somatostatin with cytosolic fraction was rapid, reversible, specific and saturable. At equilibrium, the binding of tracer was competitively inhibited by native peptide in the 1 nM to 2 µ M range of concentrations. Scatchard analysis of binding data suggested the presence of two distinct classes of somatostatin binding sites: a class with a high affinity (K_d = 7.8 ± 0.3 nM) and a low capacity (1.3 ± 0.3 pmol somatostatin/mg protein) and a class with a low affinity (K_d = 129.1 ± 2.0 nM) and a high capacity (43.5 ± 6.7 pmol somatostatin/mg protein). The binding sites were shown to be highly specific for somatostatin since neuropeptides present in cystic duct such as Leu-enkephalin, neurotensin, substance P and vasoactive intestinal peptide did practically not show competition. These findings suggest that somatostatin could contribute to the regulation of the functions of the cystic duct mucosa in physiological and pathological conditions.     

Studies on protein kinase activity and the binding of adenosine 3'5-monophosphate by membranes of hereditary spherocytosis erythrocytes.

Evidence has been presented recently of a deficiency of an endogenous membrane-associated protein kinase in erythrocytes of patients with hereditary spherocytosis (HS). We have measured endogenous protein kinase activity in erythrocyte membranes of 4 HS subjects using different membrane isolation and reaction conditions and find that the phosphorylation of the spectrin component (mean ± S.E. 17.1 ± 1.2 pmoles/10 mins per mg protein) is not significantly different to that of 4 normal controls (mean ± S.E. 20.7 ± 1.1 pmoles/10 mins per mg protein). Phosphorylation of exogenous proteins such as casein and protamine is also not deficient in HS erythrocyte membranes. Adenosine 3′5-monophosphate (cyclic AMP) binding to normal and HS erythrocyte membranes was also studied using a Millipore filtration assay. The affinity of cyclic AMP for erythrocyte membranes as determined by Hill plots of binding data from 4 HS subjects (K_D mean ± S.E. = 2.2 ± 0.2 nM) was not significantly different to 4 normal controls (K_D mean ± S.E. = 2.8 ± 0.6 nM). The rate of dissociation of bound cyclic AMP from HS membranes was also similar to control membranes. We thus cannot confirm the prediction by others that an abnormality of cyclic AMP interaction with the erythrocyte membrane underlies HS.     

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.     

Oxytocin receptors in the porcine endometrium during the estrous cycle and early pregnancy

Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using ¹²⁵I-labeled OT antagonist [d(CH₂)₅,Tyr(Me)²,Thr⁴,Tyr-NH⁹₂]-vasotocin. Binding specificity was demonstrated by displacement with four peptides related to oxytocin ([Arg⁷]-vasopressin, [Thr⁴,Gly⁷]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile³]-pressinoic acid (tocinoic acid)). The dissociation constant (K_d) of endometrial OT receptors on D0 (0.59 ± 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 ± 0.21; D15, 0.60 ± 0.14 nM; mean ± SEM). In the early luteal stage (D5), K_d (2.41 ± 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, K_d values were 3.25 ± 0.29 nM on D5 and 2.44 ± 0.44 nM on D15. Binding site concentration (B_max) on D0 (910.0 ± 25.1 fmol mg⁻¹ protein) was significantly higher than on D5 and D10 (D5, 322.5 ± 71.7; D10, 147.5 ± 25.8 fmol mg⁻¹ protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 ± 82.6; D15, 315.0 ± 20.1 fmol mg⁻¹ protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, B_max values were constant and similar to that on D5 of the early luteal stage.Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.     

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.     

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.     

Membranes with dopamine receptors coupled to G proteins: Purification from theLymnaea central nervous system

A membrane fraction, which contained dopamine receptors and heterotrimeric G proteins, was purified from homogenate of molluscan (Lymnaea) CNS tissues. Radioligand binding analysis with the use of [7.8-³H] dopamine detected the presence of a high-affinity binding site in this fraction. [7.8-³H] Dopamine was displaced in a dose-dependent manner by dopamine antagonists, S(-)-sulpiride, (±)-SKF83566, and fluphenazine. Radioligand binding analysis of purified membranes with the use of labelled GDP showed the presence of a high affinity binding site withB _max=92±5 pmol/mg of protein andK _d =64±10 nM. GDP, in contrast to GTP, markedly increased [7.8-³H] dopamine binding in the absence of metal cations (the maximum increase was 2.5-fold). Added separately, Na and Mg ions decreased the stimulatory influence of GDP. Jointly, these ions completely abolished this GDP influence on the [7.8-³H] dopamine binding. In the membrane fraction, GTPase activity in the presence of dopamine increased during an initial period and then decreased below the basal level. Therefore, we have demonstrated that in our experiments dopamine receptors in the purified membrane fraction are functionally coupled with heterotrimeric G proteins, but their interaction displays some specific features.     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.     

D2 receptor-mediated inhibition of dopamine release in the rat striatum in vitro is modulated by CB1 receptors: studies using fast cyclic voltammetry

Cannabinoid CB₁ receptors are highly expressed in the striatum where they are known to be co‐localized with dopamine D₂ receptors. There is now strong evidence that cannabinoids modulate dopamine release in the brain. Using fast cyclic voltammetry, single pulse stimulation (0.1 ms; 10 V) was applied every 5 min and peak dopamine release was measured with a carbon fibre microelectrode. Application of the D₂ receptor agonist, quinpirole, inhibited single pulse dopamine overflow in a concentration‐dependent manner (IC₅₀: 3.25 × 10^?8 M). The CB₁ receptor agonist WIN55212‐2 (WIN; 1 μM) had no effect on single pulse dopamine release (93.9 ± 6.6% at 60 min, n = 5) but attenuated the inhibitory effect of quinpirole (30 nM; quinpirole 39.0 ± 4.2% vs. quinpirole + WIN, 48.2 ± 3.7%, n = 5, p < 0.05). This affect was antagonized by the CB₁ receptor anatgonist [N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide] (AM‐251, 1 μM). Dopamine release evoked by four pulses delivered at 1 Hz (4P1Hz) and 10 pulses delivered at 5 Hz (10P5Hz) was significantly inhibited by WIN [72.3 ± 7.9% control (peak 4 to 1 ratio measurement) and 66.9 ± 3.8% control (area under the curve measurement), respectively, p < 0.05; n = 6 for both]. Prior perfusion of WIN significantly attenuated the effects of quinpirole on multiple pulse‐evoked dopamine release (4P1Hz: quinpirole, 28.4 ± 4.8% vs. WIN + quinpirole, 52.3 ± 1.2%; 10P5Hz: quinpirole, 29.5 ± 1.3% vs. WIN + quinpirole, 59.4 ±7.1%; p < 0.05 for both; n = 6). These effects were also antagonized by AM‐251 (1 μM). This is the first report demonstrating a functional, antagonistic interaction between CB₁ receptors and D₂ autoreceptors in regulating rat striatal dopamine release.     

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.     

Binding of β1-adrenoreceptor antagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial nodeantagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial node

Specific β₁-adrenoreceptors antagonist [³H]CGP 26505 binding was characterized in rat cerebral cortex and heart sinus atrial node. In both tissues [³H]CGP 26505 binding was maximal at 25°C, it was specific, saturable and protein concentration dependent. Scatchard analysis of saturation isotherms of specific [³H]CGP 26505 binding in cerebral cortex showed that [³H]CGP 26505 binds a single class of high affinity sites with a dissociation constant (K_D) of 1±0.3 nM and a maximal number of binding sites (B_max) of 40±2 fmol/mg of protein. In sinus atrial node, [³H]-CGP 26505 binds a single class of high affinity sites (K_D=1.9±0.4 nM, B_max=28±2 fmol/mg of protein).     

Dopamine receptors in bovine retina : characterization of the 3H-spiroperidol binding and its use for screening dopamine receptor affinity of drugs.

³H-spiroperidol bound in a saturable, stereospecifically displaceable manner to homogenates of bovine retina. Scatchard analysis of saturation experiments showed a K_D of 1.35 nM and a density of binding sites of 107 fmoles · mg protein^?1. Stereospecifically displaceable binding was pH and temperature dependent and linear with tissue concentration. Spiroperidol, pimozide, haloperidol and d-butaclamol were the most potent compounds in drug displacement curves (8.74 > pIC₅₀ > 7.61 M). Other neuroleptics such as cisflupenthixol, fluphenazine, clozapine, chlorpromazine and pipamperone, were one order of magnitude less potent. Among dopamine agonists, apomorphine (pIC₅₀ = 7.08 ± 0.19 M) was about 50 times more potent than dopamine itself, epinine and ADTN. Serotonin, α- and ß-adrenergic receptors agonists and antagonists were inactive. These results indicate that the sites labelled by ³H-spiroperidol in retina are dopaminergic; moreover the rank order of various antagonists and agonists observed in displacement curves suggests that this preparation could also provide a useful tool to reveal the selective affinity of drugs for the CNS dopamine receptor linked to the enzyme adenylylcyclase (D₁-receptors).     

Clozapine,a dopamine DA4 receptor antagonist,reduces gastric acid secretion and stress-induced gastric mucosal injury

Dopamine and its agonists modulate a variety of gastrointestinal functions. In light of the increasing attention directed toward novel dopamine receptors and compounds that are active at these sites, we examined the effects of a dopamine D₄ antagonist and putative antipsychotic, clozapine, in a model of conscious basal gastric acid secretion and in a model of stress-induced gastric mucosal injury. At a dose of 10.0 mg/kg i.p., clozapine significantly inhibited basal gastric acid secretion by 84% relative to vehicle. Lower doses (2.5 and 5.0 mg/kg) were inactive. Doses of 2.5, 5.0 and 7.5 mg/kg i.p. all significantly reduced restraint stress-induced gastric mucosal injury in rats. The highest dose inhibited gastric lesions by 70% relative to vehicle. We conclude that dopamine D₄ receptors, present in high concentrations in mesolimbic brain regions, modulate gastric function and pathology in addition to mesolimbic D₁ receptors, whose role in gastrointestinal function is already established.     

β-Adrenoreceptor agonists and antagonists modulate the activity of α<Subscript>2</Subscript>-adrenoreceptors in rat cortex cerebral membranes

The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective α₂-antagonist [³H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor interaction for α₂-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules to a receptor dimer. The following parameters were determined for [³H]RX821002 binding to α₂-adrenoreceptors: K _d1 = 1.57 ± 0.27 nM, B _max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α₂-adrenoreceptors was described by the same model. The affinity of α₂-adrenoreceptors for [³H]RX821002 decreased more than twofold (K _d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected with the following parameters: K _d1 = 0.61 ± 0.02 nM, K _d2 = 3.41 ± 0.13 nM, B _ml = 1.88 ± 0.028 fmol/mg of protein, B _m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α₂-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole exhibit modulating effect on the specific antagonist binding to α₂-adrenoreceptors, which results in the inhibition and alteration of [³H]RX821002 binding parameters.     

Stimulation of HCO3− secretion by the prostaglandin E2 analog enprostil: Studies in human stomach and rat duodenum

This study investigated the action of enprostil, a synthetic analog of PGE₂, on gastric HCO₃⁻ secretion in humans and on duodenal HCO₃⁻ secretion in the anesthetized rat. A previously validated 2-component model was used to calculate gastric HCO₃⁻ and H⁺ secretion in 10 human subjects. Compared to placebo, a single 70 μg oral dose of enprostil increased basal gastric HCO₃⁻ secretion from 1810 +- 340 to 3190 ± 890 μmol/hr (P < 0.05). In addition, enprostil reduced basal gastric H⁺ secretion from 5240 ± 1140 to 1680 ± 530 μmol/hr (P < 0.02). Enprostil also increased HCO₃⁻ and reduced H⁺ secretion during intravenous pentagastrin infusion. In the rat, duodenal HCO₃⁻ secretion was measured by direct titration in situ using perfused segments of duodenum just distal to the Brunner gland area dn devoid of pancreatic and biliary secretions. Addition of enprostil(10 μg/ml) to the duodenal bathing solution increased duodenal HOC₃⁻ secretion from 6.3 ± 1.3 to 15.1 ± 2.0 μmol/cm·hr (P < 0.01, n = 6). The stimulatory action of enprostil on duodenal HCO₃⁻ secretion at 10 μg/ml was comparable in magnitude and duration to that of 10 μg/ml natural PGE₂. In summary, the PGE₂ analog enprostil stimulated gastroduodenal HCO₃⁻ secretion, effects which may be beneficial in protection of the gastroduodenal mucosa against luminal acid.