Effects of cholera toxin on the coupling of thyrotropin-releasing hormone to a guanine nucleotide-binding protein in cultured GH3 cells

The effects of cholera toxin on the coupling of the thyrotropin-releasing hormone (TRH) receptor to a guanine nucleotide-binding (G) protein were examined in a GH3 clonal strain of rat pituitary tumor cells. Incubation of the cells with cholera toxin (50 ng/ml) for 16 hr caused a decrease in [3H]methyl-TRH binding to 59% of the control level and in TRH-stimulated low Km GTPase activity from 143 to 107% of the control level in the membrane-containing fraction. The effects of cholera toxin were time dependent; TRH-stimulated GTPase activity was reduced after a 3-hr incubation, whereas cholera toxin decreased [3H]methyl-TRH binding in the membrane-containing fraction after a 5-hr incubation. These results suggest that the inhibition of TRH-stimulated GTPase activity by cholera toxin treatment is not due to the decrease of receptor binding caused by this toxin. On the other hand, incubation of GH3 cell membranes with preactivated cholera toxin and NAD+ did not substantially alter the binding of [3H]methyl-TRH. In contrast, the cholera toxin-treated membranes demonstrated a partial reduction in the activity of TRH-induced low Km GTPase activity and a 10-fold increase in the concentration of guanine nucleotide required for a half-maximal effect in regulating the TRH receptor affinity for [3H]methyl-TRH. These data suggest that cholera toxin may act directly on a G protein that is associated with TRH-receptors.     

Pharmacological and biochemical comparison of thyrotropin releasing hormone (TRH) and di-methyl proline-TRH on pituitary GH3 cells.

1. The binding of [3H]-thyrotropin releasing hormone ([3H]-TRH) and [3H]-RX77368 (di-methyl proline TRH) and the ability of these peptides to stimulate phosphoinositide hydrolysis were investigated in the GH3 pituitary cell line. 2. For both peptides binding was found to be saturable with a single component (Hill slopes were, for TRH, 0.98 and for RX77368, 1.13). TRH bound with greater affinity than RX77368 Kd values were 16 nM and 144 nM respectively. Bmax values were 227 fmol mg-1 protein for TRH and 123 fmol mg-1 protein for RX77368. 3. The rank order of potency of a series of TRH analogues to inhibit binding was the same versus each peptide. However, unlike with saturation analysis, Hill slopes of all displacing ligands were less than 1.0 against both TRH and RX77368 suggesting either multiple binding sites, alteration of affinity state, negative co-operativity or some allosteric interaction. 4. Both peptides stimulated phosphoinositide hydrolysis in a dose-dependent fashion. TRH was more potent than RX77368, EC50 values were 7.9 +/- 1 nM and 96.3 +/- 3 nM respectively. 5. These in vitro data suggest that the greater in vivo potency of RX77368 is not the result of enhanced receptor affinity but is more probably due to its greater metabolic stability.     

Solubilization, characterization and partial purification of [3H]mepyramine-binding protein, a possible histamine H1 receptor, from rat liver membrane

[3H]Mepyramine binding protein, a possible subtype of histamine H1 receptors, was solubilized from rat liver membrane with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and Tween 60 as detergents and glycerol as an enhancer of solubilization. The optimal concentration of CHAPS was 10 mM and that of glycerol was 20% or more (v/v). The molecular weight of the [3H]mepyramine binding protein-detergent complex was determined to be 670K by Sepharose CL-4B gel filtration and 800K by sucrose density gradient sedimentation. By target size analysis, the molecular weights of both the membrane-bound and solubilized [3H]mepyramine binding protein were determined to be 162K. These values are similar to those of other well-characterized H1-receptor proteins, though slightly different. Simultaneous computerized analysis of the data obtained by [3H]mepyramine binding to the solubilized [3H]mepyramine binding protein indicated the presence of a single binding site with a KD value of 19.0 +/- 5.6 nM and a binding capacity (Bmax) of 6.6 +/- 2.1 pmole/mg protein. The Ki value of cold mepyramine for [3H]mepyramine binding to the solubilized receptor was 20 +/- 4 nM, whereas those of diphenhydramine, d-chlorpheniramine and triprolidine were all 2.9 +/- 0.8 microM, or about 150 times that of mepyramine. These data on the molecular and binding characteristics of the solubilized protein reported here suggest that there is a subtype of histamine H1 receptor in rat liver membrane. The solubilized preparation retained 90% and 75% of its [3H]mepyramine binding activity after storage at -80 degrees C and 4 degrees C, respectively, for 20 days. The solubilized [3H]mepyramine binding protein was purified 30-fold by Sepharose CL-4B gel filtration, Bio Gel HTP hydroxylapatite, Octyl Sepharose 4B and hydroxylapatite HPLC column chromatographies.     

Synthesis, receptor binding affinities and alpha-MSH releasing activities of TRH analogues

New syntheses of three thyrotropin releasing hormone (TRH) analogues ([Dopa2]THR, [Nic1]TRH, and [Tyr(30NO2)2]TRH) have been reported (Dopa stands for L-3,4-dihydroxyphenylalanine, Nic--for nicotinic acid and Tyr(3-NO2)--for L-3-nitrotyrosine). These three TRH analogues and five already known ones ([Aad1Tca3]TRH, [D-His2]TRH, [D-Pro3]TRH, [Pro-NH-NH2(3)]TRH and [Tyr2]TRH), were studied in vitro for their binding activity to rat pituitary TRH receptors and a-MSH releasing activity in the neuro-intermediate lobe of frogs. Competition of analogues for 3H-TRH binding to rat anterior pituitary membrane fraction was used. One of ten tested analogues ([Aad1, Tca]3 TRH) was as potent as TRH in competing for high-affinity binding sites (Kd = 8.5 nM). The binding activity of diastereoisomers ([D-His2]TRH and [D-Pro3]TRH) was reduced as well as that of analogue [Pro-NH-NH2(3)]TRH. The rest of the analogues were inactive. The binding activities were in good accordance with alpha-MSH releasing activities.     

Solubilization and preliminary characterization of mu and kappa opiate receptor subtypes from rat brain

The opiate agonists [3H]dihydromorphine (DHM, mu-selective ligand), [3H]bremazocine (potent kappa ligand), and [3H]etorphine bound stereospecifically, with high affinity, and reversibly to partially purified 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS)-solubilized extract from rat brain membranes. Recoveries of the three binding activities were as follows: [3H]DHM, 47%; [3H]bremazocine, 55%; and [3H]etorphine, 17%. Each ligand exhibited (by Scatchard analysis) binding to a class of high-affinity sites (Kd = 0.8-2 nM). Hill analyses revealed Hill coefficients of n = 1.1-1.3. Many of the properties of solubilized brain opiate receptors are similar to those of membrane-associated opiate receptors. Opiate binding in soluble fractions was inhibited by a variety of protein-modifying agents, including trypsin, proteinase K, and N-ethylmaleimide as well as by heat treatment (60 degrees, 15 min). The relative potencies of a series of opiate narcotic agonists and antagonists in displacing 2 nM [3H]etorphine binding to the CHAPS-solubilized extract was similar to that determined for rat brain homogenates. In contrast, D-Ala2, D-Leu5-enkephalin (DADLE, putative delta-selective ligand) exhibited a much lower affinity for solubilized brain opiate receptors than for the membrane-bound receptors unless assayed in the presence of manganese chloride, sodium chloride, and GTP. Mu agonist binding to solubilized receptors was inhibited relatively selectively by sodium and guanyl nucleotides. These findings lend support to the pharmacological relevance of the solubilized opiate-binding component(s). The pI of the solubilized brain opiate receptor(s) was estimated by liquid isoelectrofocusing to be pH 4. The sizes of the solubilized, prelabeled [3H]etorphine-receptor complex of the solubilized mu and kappa receptor subtypes, as assayed by stereospecific binding of [3H]DHM and [3H]bremazocine binding, respectively, were estimated by molecular exclusion chromatography. The [3H]etorphine-receptor complex migrated as a broad radioactive peak at a position corresponding to a protein of Stoke radius 63 A. A secondary peak of radioactivity was observed at the salt peak. Mu receptor activity chromatographed as two major peaks. The first of these eluted just behind, but significantly separated from, the protein void peak and corresponded to a Stokes radius of 70 A; the second eluted just ahead of the salt peak and corresponded to a radius of less than 20 A. Kappa receptor activity eluted at positions corresponding to macromolecules of 50 A and less than or equal to 20 A. Together, these findings indicate that selective mu and kappa ligands interact with high molecular weight species of somewhat different sizes as well as a lower molecular weight species, which may represent a common subunit that can bind both ligands.     

Characterization of [3H](+/-)L-364,718 binding to solubilized cholecystokinin (CCK) receptors of rat pancreas

The binding of [3H](+/-)L-364,718 (3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl )-1H-indole-2-carboxamide), an extremely potent nonpeptide cholecystokinin (CCK) receptor antagonist, to digitonin-solubilized CCK receptors from rat pancreas was characterized. [3H](+/-)L-364,718 binding to digitonin-solubilized receptors was assayed using polyethylene glycol precipitation followed by rapid filtration to separate free and bound [3H](+/-)L-364,718. Specific [3H](+/-)L-364,718 binding to solubilized receptors was dependent on the digitonin and receptor concentration and, under optimal conditions, represented greater than 90% of the total binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.53 nM and a Bmax of 3.1 pmol/mg protein. Specific [3H](+/-)L-364,718 binding to solubilized CCK receptors was inhibited by both CCK receptor agonists and antagonists in a stereospecific manner. After solubilization, the affinities of various antagonists to displace specific [3H](+/-)L-364,718 binding were similar to those obtained with membrane-bound receptors; however, the affinities of CCK agonists were reduced 10-100 times. Collectively, the data presented indicate that [3H](+/-)L-364,718 represents a new antagonist ligand which has apparent advantages over the agonist ligand [125I]CCK in assaying digitonin-solubilized receptors. Gel filtration of the digitonin-solubilized CCK receptors followed by [3H](+/-)L-364,718 binding determinations revealed an estimated molecular weight of 400,000 daltons.     

Solubilization, purification, and functional reconstitution of 5-hydroxytryptamine3 receptors from N1E-115 neuroblastoma cells.

5-Hydroxytryptamine3 (5-HT3) receptors from N1E-115 neuroblastoma cells were solubilized using 1.1% n-octylglucoside; five other detergents were less effective. Purification was achieved by affinity chromatography using immobilized GR119566X and biospecific elution with quipazine. Saturation analyses with [3H] GR67330 binding revealed high affinity binding to homogeneous populations of sites in both the solubilized (Kd = 0.05 +/- 0.02 nM) and purified (Kd = 0.10 +/- 0.04 nM) preparations. Competition experiments indicated that the solubilized and purified receptor preparations retained the characteristics observed in N1E-115 cells in vivo. Polyacrylamide gel electrophoresis of the purified receptor revealed a single protein band of 54.7 +/- 1.3 kDa. The purified receptor was incorporated into liposomes, and the functional integrity of the protein was demonstrated by measurement of m-chlorophenylbiguanide-stimulated 22Na uptake. Saturation analysis of the reconstituted preparation revealed a Kd of 0.24 +/- 0.07 nM and suggested that 0.2% of 5-HT3 receptors present in the original membrane preparation had been incorporated into liposomes.     

Characterization and autoradiographic distribution of [3H]AF-DX 384 binding to putative muscarinic M2 receptors in the rat brain.

The novel radioligand [3H]AF-DX 384 binds specifically and saturably to putative muscarinic M2 receptor sites in homogenates of rat cerebral cortex. In saturation studies, [3H]AF-DX 384 appears to bind to two subpopulations of sites/states, one of high affinity (Kd1 = 0.28 +/- 0.08 nM) and another of low affinity (Kd2 = 28.0 +/- 5.0 nM). The maximal binding capacity (Bmax) of [3H]AF-DX 384 binding sites represented 9.7 +/- 2.3 fmol/mg protein (Bmax1) and 1993 +/- 551 fmol/mg protein (Bmax2) for the high and low affinity sites/states, respectively. The ligand selectivity profile of [3H]AF-DX 384 (at 2 nM) revealed that (-)-quinuclidinyl benzylate = atropine greater than 4-diphenylacetoxy-N-methylpiperidine methobromide greater than AQ-RA 741 greater than AF-DX 384 greater than UH-AH 371 much greater than methoctramine greater than oxotremorine-M greater than hexahydro-sila-defenidol much greater than pirenzepine greater than carbamylcholine much much greater than nicotine. This suggests that under our assay conditions [3H]AF-DX 384 binds mostly to M2-like muscarinic receptors in the rat central nervous system. This is further supported by the clear M2-like pattern of distribution observed using quantitative receptor autoradiography. High densities of specific labelling were seen in areas such as the hypoglossal nucleus, the pontine nucleus, the superior colliculus, the motor trigeminal nucleus, various thalamic nuclei and certain cortical laminae. Compared to [3H]AF-DX 116, the percentage of specific binding detected with [3H]AF-DX 384 was much higher. This is likely to be related to the greater chemical stability and affinity of [3H]AF-EX 384. In addition, autoradiograms obtained with [3H]AF-DX 384 (2 nM) are of better quality with film exposure periods five shorter than those needed for [3H]AF-DX 116 (10 nM). Therefore, [3H]AF-DX 384 displays a good selectivity for muscarinic M2 sites and offers major advantages, including higher affinity and greater stability, over previously used ligands.     

Comparison of CCK-8 receptors in the pancreas and brain of rats using CCK-8 analogues

The characteristics of cholecystokinin (CCK) receptors in rat pancreatic acini and in various regions of the brain were examined using synthetic CCK-8 or CCK-7 analogues. 3H-propionylated CCK-8 [( 3H]CCK-8) was used as a ligand. 1) The pancreatic CCK receptor had a single high affinity binding component with a dissociation constant, Kd, of 0.76 nM and a maximum number of specific binding sites, Bmax, of 271.91 fmol/mg protein. On the other hand, the CCK receptor in the cerebral cortex had a Kd of 1.66 nM and a Bmax of 30.15 fmol/mg protein. 2) The order of the potencies of CCK-7 and CCK-8 analogues with a substitution at position 3 or 4 to displace [3H]CCK-8 specific binding to the pancreatic acini was as follows: CCK-8 greater than CCK-7 = SucCCK-7 greater than Suc[Sar3]CCK-7 greater than Suc[D-Trp3]CCK-7 greater than Suc[D-Ala3]CCK-7 greater than [D-Trp4]CCK-8 = [D-Ala4] CCK-8. This order of potencies of CCK analogues was greatly different from that in the cerebral cortex. 3) The carboxy-terminal tetra-peptide (CCK-4) and penta-peptide (CCK-5) had very weak potencies in displacing [3H]CCK-8 binding in the pancreatic acini, which were 20 to 30-fold less than their potencies in the cerebral cortex. These results suggest that the recognition sites for CCK analogues in the pancreatic and brain CCK receptors are different.     

Beta-adrenergic receptors in guinea-pig liver plasma membranes and thermal lability of [3H]dihydroalprenolol binding sites

beta-Adrenergic receptors in guinea-pig liver plasma membranes were characterized by radioligand binding, using l-[3H]dihydroalprenolol ([3H]DHA), l-3-[125I]iodocyanopindolol ([125I]CYP) and dl-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2- one hydrochloride [( 3H]CGP-12177). The binding of both [125I]CYP and [3H]CGP-12177 to membranes exhibited high affinity (Kd = 3.5 +/- 0.2 pM for [125I]CYP and 0.75 +/- 0.10 nM for [3H]CGP-12177) and stereospecificity; the maximal binding sites were 130 +/- 15 and 137 +/- 8 fmoles/mg protein respectively. Catecholaminergic agonists competed for these binding sites in the order l-isoproterenol greater than l-epinephrine greater than l-norepinephrine, which is typical for beta 2-adrenergic receptors. The binding data are supported by parallel experiments on adenylate cyclase activation by catecholamines, and on antagonism of this activation by beta 1- and beta 2-selective blockers. The binding of [3H]DHA was excessive (Bmax = 21.4 pmoles/mg protein), exhibited low affinity (Kd = 34.6 nM), and lacked stereospecificity. When liver membranes were incubated at 50 degrees for 40 min in the presence of an agonist, l-isoproterenol, the binding of [3H]DHA to the heat-treated membranes exhibited high affinity (Kd = 1.07 +/- 0.17 nM) and the Bmax was reduced to 139 +/- 22 fmoles/mg protein. In such membranes, as opposed to native membranes, stereospecificity was evident and catecholaminergic agonists competed for the binding sites in the order typical for beta 2-adrenergic receptors. However, agonist competition of the binding to the heat-treated membranes could not be modulated by guanine nucleotides, indicating a loss of communication between the receptor and the guanine nucleotide regulatory protein.     

Diversity of thyrotropin-releasing hormone receptors in the pituitary and discrete brain regions of rats

In order to analyze the receptor properties of central nervous system (CNS)-stimulant thyrotropin-releasing hormone (L-pyroglutamyl-L-histidyl-L-prolinamide, TRH), we evaluated the binding of TRH and its analog taltirelin hydrate ((-)-N-[(S)-hexahydro-1-methyl-2,6-dioxo-4-pyrimidinylcarbonyl]-L- histidyl-L-prolinamide tetrahydrate; taltirelin, TA-0910) in rat anterior pituitary and several brain regions. There was a specific binding of [3H]methyl TRH (MeTRH) in the anterior pituitary, hypothalamus, brain stem, cerebral cortex and cerebellum with Kd values of 1.0-1.6 nM. The inhibition of [3H]MeTRH binding by TRH and taltirelin was monophasic in the anterior pituitary, hypothalamus and brain stem with Ki values of 6.3-8.0 nM and 145.5-170.4 nM for TRH and taltirelin, respectively. In contrast, the biphasic inhibition was revealed in the cerebral cortex and cerebellum. The Ki values for TRH and taltirelin were 4.1-4.3 nM and 67.8-73.4 nM for the high affinity binding site and 3.6-4.2 microM and 82.3-197.5 microM for the low affinity binding site, respectively. Addition of 100 microM GTP or its analog 5'-guanylylimidodiphosphate (Gpp[NH]p) affected neither the biphasic inhibition by TRH nor that by taltirelin. Thus the results suggest the presence of distinct high and low affinity TRH receptors in the CNS in contrast to the pituitary.     

Characterization of alpha 2-adrenergic receptors of calf retina membranes by [3H]-rauwolscine and [3H]-RX 781094 binding

Alpha 2-adrenergic receptors were identified in calf retina membranes by binding of the radiolabelled antagonists [3H]-RX 781094 and [3H]-rauwolscine. When 10 microM phentolamine was used to determine the non-specific binding, both radioligands labelled a single class of non-cooperative sites: Bmax = 1051 +/- 252 fmol/mg protein, Kd = 5.1 +/- 1.5 nM for [3H]-RX 78104 and Bmax = 1167 +/- 449 fmol/mg protein, Kd = 21.0 +/- 4.1 nM for [3H]-rauwolscine. Competition binding experiments showed the typical pharmacological potency order of alpha 2-adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin. Agonist competition binding curves revealed the presence of two receptor populations, having respectively high affinity (70% of the total receptor population) and low affinity for agonists, but with the same affinity for the antagonists. The high affinity sites could be converted into low affinity sites by guanine nucleotides. The non-specific binding of [3H]-RX 781094 was the same if 0.1 mM (-)-epinephrine was used instead of phentolamine. In contrast, the non-specific binding of [3H]-rauwolscine was markedly lower with (-)-epinephrine than with phentolamine. Under this condition, the Scatchard plot of [3H]-rauwolscine saturation binding was curvilinear, indicating the presence of low affinity sites for the radioligand in addition to alpha 2-adrenergic receptors. Competition binding experiments revealed that these low affinity sites were distinct from adrenergic receptors: the catecholamine agonists (-)- and (-)-epinephrine, (-)-norepinephrine, (-)-isoproterenol and dopamine competed with similar Ki values (microM range) whereas clonidine did not interact. Furthermore, these sites bound reserpine and the alpha 2-adrenergic antagonists yohimbine and rauwolscine but not phentolamine.     

Identification of drug receptors in porcine dental pulp by the radioligand binding assay

1. The acetylcholine (ACh), histamine and serotonin (5-HT) receptors in porcine dental pulp were characterized by the radioligand binding assay. 2. For [3H]nicotine binding site, Kd was 8.06 +/- 1.65 nM and Bmax was 270.83 +/- 32.68 fmol/mg protein. 3. For [3H]QNB binding site, Kd was 1.04 +/- 0.14 nM and Bmax was 24.83 +/- 3.09 fmol/mg protein. 4. For [3H]histamine binding site, Kd was 1.22 +/- 0.1 nM and Bmax was 283.15 +/- 33.1 fmol/mg protein. 5. For [3H]5-HT binding site, Kd was 1.41 +/- 0.1 nM and Bmax was 53.1 +/- 3.4 fmol/mg protein. 6. These findings indicate that the specific receptors for ACh, histamine and 5-HT are present in the porcine dental pulp, and that the ACh receptor is predominantly nicotinic.     

Studies on kinetics of [3H]beta-funaltrexamine binding to mu opioid receptor

beta-Funaltrexamine (beta-FNA) was shown to be a reversible kappa agonist and an irreversible mu antagonist. [3H]beta-FNA at low concentrations (less than 10 nM) covalently binds to mu but not delta or kappa opioid receptors in brain membranes. The interaction between beta-FNA and mu opioid receptors was thought to involve two steps; a reversible ligand-receptor complex is formed before the formation of an irreversible complex, based on observations in bioassays in vitro. In this study, we investigated whether such a two-step process occurred in binding using bovine striatal membranes and determined the kinetic parameters by examining the time courses of both reversible and irreversible binding of [3H]beta-FNA to mu opioid receptors. Specific binding was defined as the difference between binding in the presence of levorphanol and dextrorphan (1 microM). Reversible binding was determined as the difference between membrane (reversible and irreversible) binding and irreversible binding. At 25 degrees, the rate of formation of irreversible [3H]beta-FNA-receptor complex increased as the concentration increased and reached a plateau at 2 nM; further increase in [3H]beta-FNA concentration did not enhance the rate of formation, indicating that the rate saturation effect exists for irreversible binding of [3H]beta-FNA to mu opioid receptors. At 10 degrees and low concentrations (less than 1 nM) of [3H]beta-FNA, appreciable reversible binding to opioid receptors occurred before any irreversible [3H]beta-FNA-receptor complex could be detected. These observations support the notion that reversible binding occurs before alkylation of the receptor. The binding of [3H]beta-FNA to mu opioid receptors was thus modeled to allow for such a two-step process: (formula; see text) A mathematical analysis method was derived to allow determination of all kinetic parameters (k+1, k-1, k2, and Kd) of such a two-step reaction. Values of k2, k+1, k-1, and Kd were determined at 10 degrees for 0.5, 0.25, and 0.125 nM [3H]beta-FNA and were found to be very similar among these three concentrations. Raising the incubation temperature from 10 degrees to 37 degrees greatly enhanced the values of k+1, k-1, and k2 without affecting Kd. At 37 degrees incubation without 200 mM NaCl significantly decreased the values of k+1, k-1, and k2 without affecting Kd. NaCl increased the irreversible binding, probably by shifting the equilibrium towards a conformation that binds more easily with beta-FNA. Under all conditions examined, the value of k-1 was found to be at least 5-fold greater than k2, indicating that the majority of the reversible complex dissociates and only a small portion proceeds to form irreversible complex. This finding is consistent with published observations that only a portion of beta-FNA binding to mu opioid receptors is irreversible. In conclusion, [3H]beta-FNA binds reversibly to mu opioid receptors before forming covalent bonding...     

Direct labeling of rat M3-muscarinic receptors by [3H]4DAMP

The muscarinic receptors of rat submaxillary gland, rat heart and rat cortex were directly labeled using the ligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide [( 3H]4DAMP). In the rat submaxillary gland, [3H]4DAMP predominantly bound with high affinity (Kd = 0.2 nM) to a population of binding sites that displayed the pharmacology of the M3 muscarinic receptor subtype. In rat heart, [3H]4DAMP labeled the M2 muscarinic receptor with low affinity (Kd = 4 nM). In rat cortex [3H]4DAMP predominantly bound to a population of sites with high affinity (Kd = 0.2 nM). The pharmacology of these sites was consistent with [3H]4DAMP labeling both M1 and M3 muscarinic receptors present in rat cortex with high affinity. These data indicate that [3H]4DAMP represents a useful ligand for selectively labeling the M1 and M3 muscarinic receptor subtypes.     

Characterization of adenosine receptors in brush-border membranes from pig kidney.

1. The adenosine receptors from pig kidney proximal tubules have been studied in membrane vesicle preparations derived from either luminal (brush-border membranes-BBM-) or basolateral (BL) sides. There was a substantial amount of A2-like NECA binding in both preparations, but the A1 subtype of adenosine receptors was not found in either BBM or BL membranes. The use of [3H]-CGS21680 which is a more specific ligand for A2a receptors revealed true adenosine receptors in the BBM. 2. The kinetic parameters for [3H]-CGS21680 binding to pig renal BBM were: Bmax = 1.48 pmol mg-1 protein and Kd = 150 nM. In the presence of Gpp(NH)p the affinity decreased (Kd = 220 nM), whereas the addition of Mg2+ induced a marked increase in affinity (Kd = 83 nM). These equilibrium constants are higher than those found for the A2a adenosine receptors present in pig brain striatal membranes (Kd = 12 nM), and are close to those found in rat renal BBM (Kd = 90 nM). 3. The order of potency of agonist and antagonists was not consistent with the presence of either A1 or A2 receptors, but it was very similar to the agonist order of potency for the A3 receptor subtype. Furthermore, the blockade of the [3H]-CGS21680 binding by both cholera and pertussis toxin further supports the view that the subtypes present in BBM are neither A1 nor A2. 4. Overall the results suggest the presence in BBM of an A3 receptor, or of a new subtype of adenosine receptor, which is linked to G proteins sensitive to both cholera and pertussis toxins.     

Protein kinase C inhibits TRH-stimulated phosphoinositide hydrolysis in GH3 cells

The GH3 pituitary tumor line expresses TRH receptors that stimulate phosphoinositide hydrolysis and hormone secretion. After protein kinase C was identified in GH3 cells by direct labeling with [3H]phorbol dibutyrate (PDB), the response to phorbol ester and TRH pretreatment on subsequent TRH-stimulated inositol phosphate (IP) accumulation was found to be inhibitory. Both phorbol myristate acetate (PMA) and PDB were effective in this regard at low nM concentrations within a few minutes, whereas phorbols that do not stimulate protein kinase C were without effect. Furthermore, the mono-, bis- and tris-phosphate forms of IP were all reduced by an average of 30-40% after 5 min of PMA. TRH concentration-response studies indicated a clear change in TRH efficacy induced by PMA. Finally, preincubation with TRH itself was also capable of reducing the subsequent response to TRH. Because TRH receptor action is thought to activate protein kinase C by producing diacylglycerol, these data indicate a negative feedback system via protein kinase C operative during continuous exposure to TRH in GH3 cells.     

Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid [3H]D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid [3H]U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by [3H]U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan [3H]ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, [3H]ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of [3H]ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with [3H]U69593. Saturation studies using the nonselective opioid [3H]bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue). The total binding capacity obtained with [3H]bremazocine (Rtotal = 19.1 pmol/g of tissue) was different from either the sum of the binding capacities of mu-, delta, and kappa-selective ligands or the binding capacity of [3H]ethylketazocine obtained under unsuppressed conditions. These results suggest that [3H]bremazocine labels additional opioid sites, namely the kappa 2 receptors, in contrast to kappa 1 sites labeled with [3H]U69593. In experimental conditions where the binding of [3H]bremazocine at mu, delta, and kappa 1 receptors was quenched by selective blockers, [3H]bremazocine recognizes the kappa 2 receptors with one homogeneous affinity state (Kd = 3.45 nM, R = 8.23 pmol/g of tissue). However, competition studies suggest that some opioids bind to these kappa 2 receptors with heterogeneous affinity states (high and low affinity states), whereas others bind with one apparently homogeneous affinity state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic receptor subtypes in rat pancreatic islets: binding and functional studies

Cholinergic agents are potent modulators of insulin release that act via muscarinic receptors. We now investigated the muscarinic receptor subtype present in rat pancreatic islets in binding and functional studies. Binding of 5 nM [3H]N-methylscopolamine ([3H]NMS) was half maximal at 30 min. At 60 min, the maximal total binding was 1.29% and the non-specific binding (presence of 100 microM atropine) was 0.18% of the total radioactivity per 10 micrograms islet protein. Unlabelled atropine inhibited [3H]NMS binding with an IC50 of ca. 30 nM. The rank order of antagonist high-affinity binding was atropine greater than sila-hexocyclium methyl sulfate (SiHC; M1 greater than M3 greater than M2) greater than pirenzepine (M1 greater than M2 approximately M3) = methoctramine (M2 greater than M1 greater than M3). The high-affinity Kds were 8.5, 56, 1300 and 1300 nM, respectively. The high affinity Kd of the muscarinic receptor agonist, arecaidine propargyl ester (APE), was 8.1 nM. The EC50 for the biological effects of APE on insulin and glucagon secretion was 3.2 and 2.3 nM. The rank order for the high-affinity biological effects of antagonists (inhibition of APE-mediated insulin/glucagon release) was almost the same as for binding. The data indicate that rat pancreatic islets contain neither an M1 subtype (high-affinity for pirenzepine) nor an M2 subtype (high-affinity for methoctramine) receptor. However, the data evidence an M3 receptor subtype, since SiHC in the absence of the M1 receptor subtype shows a relatively high affinity to the receptors in rat pancreatic islets.     

Chemical modification and irreversible inhibition of striatal A2a adenosine receptors.

The ligand recognition site of A2a-adenosine receptors in rabbit striatal membranes was probed using non-site-directed labeling reagents and specific affinity labels. Exposure of membranes to diethylpyrocarbonate at a concentration of 2.5 mM, followed by washing, was found to inhibit the binding of [3H]CGS 21680 and [3H]xanthine amine congener to A2a receptors, by 86 and 30%, respectively. Protection from diethylpyrocarbonate inactivation by an adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine, and an antagonist, theophylline, suggested the presence of two histidyl residues on the receptor, one associated with agonist binding and the other with antagonist binding. Binding of [3H]CGS 21680 or [3H]xanthine amine congener was partially restored after incubation with 250 mM hydroxylamine, further supporting histidine as the modification site. Preincubation with disulfide-reactive reagents, dithiothreitol or sodium dithionite, at greater than 5 mM inhibited radioligand binding, indicating the presence of essential disulfide bridges in A2a receptors, whereas the concentration of mercaptoethanol required to inhibit binding was greater than 50 mM. A number of isothiocyanate-bearing affinity labels derived from the A2a-selective agonist 2-[(2-aminoethylamino) carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC) were synthesized and found to inhibit A2a receptor binding in rabbit and bovine striatal membranes. Binding to rabbit A1 receptors was not inhibited. Preincubation with the affinity label 4-isothiocyanatophenylaminothiocarbonyl-APEC (100 nM) diminished the Bmax for [3H]CGS 21680 binding by 71%, and the Kd was unaffected, suggesting a direct modification of the ligand binding site. Reversal of 4-isothiocyanatophenylaminothiocarbonyl-APEC inhibition of [3H]CGS 21680 binding with hydroxylamine suggested that the site of modification by the isothiocyanate is a cysteine residue. A bromoacetyl derivative of APEC was ineffective as an affinity label at submicromolar concentrations.