Localisation of [3H] clonidine binding in rat neurohypophysis by means of electron-microscopic autoradiography

Summary Electron-microscopic autoradiography of rat neurohypophyses treated with [³H] clonidine, an ₂-agonist, showed that binding apparently occurred preferentially at the neurosecretory endings and blood vessels rather than on the pituicytes. Since it is known that clonidine has a high affinity for plasma proteins, the distribution over the neurosecretory nerve endings would suggest the existence of presynaptic ₂-binding sites on neurosecretory neurones, which could indicate a regulatory function for catecholamines in neurohypophysial hormone release.     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin.

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na+ + K+)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.     

[3H]ouabain autoradiography of frog retina

The kinetics and distribution of ouabain binding in retinas of Rana pipiens were examined quantitatively by scintillation counting and freeze-dry autoradiography. The time-course of binding at several concentrations was consistent with a bimolecular reaction. Estimated equilibrium binding levels gave a Michaelis-Menton relationship with a Km = 8.3 x 10(-8) M and a maximum binding level (Bmax) = 4.4 x 10(-8) mol/g protein. The distribution of binding sites measured autoradiographically varied considerably between layers. The photoreceptor, inner plexiform, and optic nerve fiber layers exhibited the heaviest binding. Within the photoreceptor layer, binding was nonuniform. Binding in the outer segment decreased distally, averaging approximately 4% of that in the proximal receptor layers (Bmax = 4.6 x 10(-6) M). The origin of the outer segment activity is uncertain at light microscope resolution, as it may be a result of inner segment calyceal processes. Binding within the proximal receptor layers was also nonuniform. Several peaks were observed, with those at the inner segment and synaptic layers being especially noticeable. Assuming an absence of glial cell binding in the proximal receptor layers, we calculated there to be 13 x 10(6) ouabain or Na+,K+ pump sites per rod receptor. Limited measurements suggest a Bmax of approximately 8 x 10(-6) M for the inner plexiform layer.     

Muscarinic receptors in the prenatal mouse embryo. Comparison of M35-immunohistochemistry with [3H]quinuclidinyl benzylate autoradiography

Muscarinic cholinergic receptors are widespread in nervous tissue and smooth muscsle or paracrine epithelial cells of various organs. In the embryo, muscarinic receptors are transitorily expressed in the early blastoderm and later on in blastemic tissues during morphogenesis. Recently, a monoclonal antibody (M35) against muscarinic receptor from calf brain became available. In the present study the use of M35-immunohistochemistry is compared to autoradiographic localization of muscarinic binding sites in the mouse embryo. The aim of the study is to test the suitability of the antibody for localization of muscrinic receptors in embryonic tissues. For autoradiography whole-body sagittal cryostat sections of the 17- and 18-day mouse embryo were covered with LKB-Ultrofilm after incubation with the radioactive ligand [³H] quinuclidinyl benzylate (QNB). For immunohistochemistry cryostat sections of formalin fixed tissues were used. In general, all tissues exhibiting ligand binding were also recognized by the antibody. M35-immunohistochemistry resulted in higher spatial resolution of receptor localization than [³H]QNB autoradiography. Definitive muscarinic receptors were observed in smooth muscle and the epithelial lining of the vascular, intestinal, respiratory and urinary system, in the brain, spinal cord and peripheral nerves. The embryonic type of the muscarinic receptor was detected in the mesothelium of lung and liver, in the nephrogenic blastema of the metanephros, and in lung mesenchyme. A large amount of embryonic muscarinic receptors was found in the remnants of the notochord and in the nucleus pulposus of the developing vertebral column. A function in morphogenesis is discussed of the embryonic muscarinic receptor.     

Reconstitution of the type II [3H]estradiol binding site with recombinant histone H4

Previously, we identified the rat uterine nuclear type II [3H]estradiol binding site as histone H4 and an unknown 35 kDa protein with histone H4 immunoreactivity. Studies using calf thymus histones indicated that the 35 kDa protein was likely a dimer of histone H3 and H4. Further study of the type II site required methodology for producing sufficient quantities of recombinant histones, which retained ligand-binding properties. A variety of production methods produce sufficient quantities of histone for binding analyses were evaluated prior to finding a successful technique. The present studies describe techniques for the production of recombinant histones that retain the ligand binding properties of type II binding site. Binding studies with recombinant protein mirrored [3H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [3H]estradiol with a low affinity (Kd approximately 20 nM) and in a cooperative fashion (curvilinear Scatchard plot; Hill coefficient approximately 4). Although histone H3 does not appear to bind ligand, regeneration of the histone H3/H4 pair produced a 35 kDa protein equivalent to the 35 kDa protein labeled with [3H]luteolin in rat uterine nuclear extracts and calf thymus histones. These data confirm the identification of histone H4 as a key component of the type II site. Future studies with recombinant proteins will lead to the identification of the "nucleosomal ligand-binding domain" for methyl-p-hydroxyphenyllactate (MeHPLA) and related ligands and delineation of their epigenetic control of gene expression and cell proliferation.     

Alpha adrenergic drugs inhibit [3H]-QNB binding to muscarinic receptors of rat heart, brain and parotid gland membranes

Alpha adrenergic agonists and antagonists as clonidine, guanfacine, yohimbine, phenylephrine and prazosin inhibited the [3H]-QNB binding to rat brain cortex muscarinic acetylcholine receptor (mAChR, M-1 subtype), heart (M-2 subtype) and parotid gland homogenate (M-3 subtype) in a dose-dependent competitive fashion. Ki values were between 10(-6) and 10(-3) M. Hill coefficients were about 1. No correlation was found between mAChR inhibiting capacity of these drugs and their activity on alpha adrenergic receptors. In contrast, other transmitters, as dopamine, GABA, glutamic acid, histamine, serotonin, isoproterenol and platelet activating factor (PAF) did not affect the QNB binding.     

Diffusion of [3H]dexamethasone in rat subcutaneous slices after injection measured by digital autoradiography

A relatively simple method for the determination of the diffusion coefficient of a substance that has been injected into tissue is described. We illustrate this method using [3]dexamethasone injected into the subcutaneous tissue of rats. Digital autoradiography was used to measure the distribution of the [3H] dexamethasone within the subcutaneous tissue at 2.5 and 20 min after injection. Measured concentration profiles of the injection were compared to a mathematical model of drug diffusion from an injection. There was good agreement between the experimental data and the mathematical model. The diffusion coefficient found using this simple injection method was (4.01 +/- 2.01) x 10(-10) m2/s. This D value was very close to the value of D = (4.11 +/- 1.77) x 10(-10) m2/s found previously using different mathematical and experimental techniques with osmotic pumps implanted for 6, 24, and 60 h in rats (1). The simple method given here for the determination of the diffusion coefficient is general enough to be applied to other substances and tissues as well.     

Regional heterogeneity of rat brain phencyclidine (PCP) receptors revealed by photoaffinity labeling with [3H] azido phencyclidine

Photoaffinity labeling of rat brain phencyclidine (PCP) receptors with [3H] azido phencyclidine ([3H]AZ-PCP) reveals the existence of five polypeptides which are specifically labeled by the affinity probe (Mr's 90,000, 62,000, 49,000, 40,000 and 33,000). These labeled components are unevenly distributed in rat brain. In the frontal cortex, thalamus and olfactory bulb, the major bands labeled are the Mr's 90 K and 62 K polypeptides; in the cerebellum most of the labeling is in the 90 K and 33 K bands; and in the hippocampus all but the Mr 40 K band are heavily labeled. Together with dexoxadrol/[3H]PCP competition binding data, which indicated the existence of high and low affinity dexoxadrol/PCP binding sites, these results suggest regional heterogeneity of PCP receptors. The regional distribution of the high affinity dexoxadrol binding sites correlates best with that of the Mr 90 K polypeptide.     

Quantitative autoradiography of [3H]CTOP binding to mu opioid receptors in rat brain

[3H]H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4 nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69, 593 were very weak inhibitors of [3H]CTOP binding (IC50 values of 234-3631 nM). Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density (nucleus of the solitary tract, clusters in the caudate-putamen, interpeduncular nucleus, superior and inferior colliculus, subiculum, substantia nigra zona reticulata, medial geniculate, locus coeruleus and dorsal motor nucleus of the vagus) and regions of moderate labeling (areas outside of clusters in the caudate-putamen, cingulate cortex, claustrum and nucleus accumbens). The cerebral cortex (parietal) showed a low density of [3H]CTOP binding.     

Brain receptor autoradiography with [3H]-YM 09151-2: a ligand for labeling dopamine D-2 receptors

Using the technique of in vitro receptor autoradiography to slide-mounted tissue sections, we studied the suitability of [3H]-YM-09151-2 as a ligand for labeling D-2 receptors in adult F344 rat brains. Specific [3H]-YM-09151-2 binding accounted for 70-80% of the total bound ligand and reached equilibrium after a 60-90 minute incubation. Scatchard analysis revealed a Kd of 626 pM. The apparent Bmax was 23.2 fmol/tissue section. Autoradiographs demonstrated high grain densities in the striatum and olfactory tubercle. Diffuse specific binding was also observed in the cortex.     

Inhibition by theanine of binding of [3H]AMPA, [3H]kainate,and [3H]MDL 105,519 to glutamate receptors

In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.     

Composition and labeling by [3H]glycerol and [3H]serine of phospholipids of the chicken retina and optic tectum

The content and fatty acid composition of phospholipids and the in vivo labeling of lipids by [3H]glycerol and [3H]serine was studied in the retina and the optic tectum of young chickens. The tectum had a higher content of phospholipids and a significantly lower ratio of choline (CGP) to ethanolamine (EGP) glycerophospholipids than the retina. Lipids of the chicken optic system were characterized by a high proportion of polyenoic fatty acids of the n-6 series compared to other species. Intravitreally injected [3H]glycerol was incorporated into all glycerol-containing lipids of the retina, especially in CGP and EGP. Most of the label from [3H]serine was found in serine glycerophospholipids (SGP). The time-dependent distribution of both precursors among retinal lipids was consistent with de novo synthesis as well as metabolic interconversions of lipids. Thus, [3H] from serine also appeared in EGP and CGP, indicating the presence and activity of SGP decarboxylase and EGP-n-methyl transferase. Lipids labeled with both precursors in retina were subsequently found in the tectum, via axoplasmic transport. Even though different lipid classes were labelled by each precursor the proportion of lipids transported to the tectum was similar in both cases (about 1% of the label present in retina).     

3H]epinephrine and [3H]norepinephrine binding to alpha-noradrenergic.

(±)-[³H]Epinephrine and (?)-[³H]norepinephrine bind saturably to calf cerebral cortex membranes under appropriate incubation conditions in a fashion indicating that they label α-noradrenergic receptors. Binding of the two [³H]catecholamines is saturable with dissociation constants of 20–30 nM. Binding is stereoselective with (?)-norepinephrine displaying about twenty times greater affinity than (+)-norepinephrine. The relative potencies of catecholamines in competing for these binding sites parallels their relative pharmacologic effects at α-noradrenergic receptors in numerous tissues. Thus, (?)-epinephrine is 2–3 times more potent than (?)-norepinephrine and 500 times more potent than (?)-isoproterenol. Binding is inhibited by low concentrations of the α-antagonists phentolamine and phenoxybenzamine but not by the β-antagonist propranolol.     

Visualization of the NMDA recognition site in rat and mouse spinal cord by [3H]CGS 19755in vitro autoradiography

Summary The possibility to visualize the NMDA recognition site with [³H]CGS 19755in vitro autoradiography was evaluated in rat brain and spinal cord sections and thereafter used to study the distribution of the NMDA recognition site in rat and mouse spinal cord. The [³H]CGS 19755 binding was concentrated to the dorsal horn, in particular to the substantia gelatinosa. Notable binding was also seen in the intermediate area and ventral horn, while some binding was observed in the funiculi. No major differences were seen in [³H]CGS 19755 binding at various levels of the rat or mouse spinal cord, although a more dense binding was seen in the mouse. A similar distribution of the [³H]CGS 19755 specific binding and the NMDA receptor associated ion-channel site, labeled with [³H]TCP, was found in the mouse spinal cord. Taken together, our data indicate that the NMDA recognition site can be visualized in rat and mouse spinal cord byin vitro [³H]CGS 19755 autoradiography.Abbreviations NMDA N-methyl-D-aspartate - CGS 19755 Cis-4-phosphonomethyl-2-piperidine carboxylic acid - D-AP5 D(—)-2-Amino-5-phosphonopentanoic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine - MK-801 (±)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate - AMPA -Amino-3-hydroxy-5-methyl-isoxazolepropionic acid - kainate 2-Carboxy-3-carboxymethyl-4-isopropenyl pyrrolidine - CGP 39653 D,L-(E)-2-amino-4-propyl-5-phosphonopentenoic acid     

An improved method for labeling carboxyatractyloside by [3H]KBH4

Carboxyatractyloside was labeled with [3H]KBH4 after oxidation of the primary alcohol of the glucose disulfate moiety by dicyclohexylcarbodiimide and P2O5 under anhydrous conditions in a dimethylsulfoxide medium. The 3H-labeled product was purified by DE 52 column chromatography followed by Cellulofine GCL 25 column chromatography. The final 3H-labeled product gave a single spot on a thin-layer chromatogram, and its Rf value was the same as that of authentic carboxyatractyloside. The biological activities (such as inhibition of state 3 respiration and binding to the adenine nucleotide carrier) were also comparable with those of authentic carboxyatractyloside.     

Hepatic alpha-adrenoreceptor: specific and irreversible labeling by [3H]-phenoxybenzamine.

The binding of [³H]-phenoxybenzamine, a potent irreversible alpha-adrenergic antagonist, to alpha-adrenergic binding sites in rat liver plasma membranes was a saturable process with a number of binding sites of 1600 fmol/mg of membrane protein. No significant dissociation of this ligand occurred after dilution for 2 hours, or repeated washing of the labeled membranes. The specificity of the binding site was characterized by an order of potency of various drugs typical for an alpha-adrenergic receptor. Binding showed strict stereospecificity since R(+)phenoxybenzamine exhibited a two order of magnitude higher affinity than its S(?)isomer. A SH group was involved in the binding of both [³H]-phenoxybenzamine and [³H]-dihydroergocryptine to alpha-receptor. It appears therefore that [³H]-phenoxybenzamine binds irreversibly and specifically to the alpha-adrenoreceptor and that it might prove adequate in the solubilization and purification of the alpha-adrenoreceptor.     

Class I HDAC imaging using [3H]CI-994 autoradiography

[³H]CI-994, a radioactive isotopologue of the benzamide CI-994, a class I histone deacetylase inhibitor (HDACi), was evaluated as an autoradiography probe for ex vivo labeling and localizing of class I HDAC (isoforms 1–3) in the rodent brain. After protocol optimization, up to 80% of total binding was attributed to specific binding. Notably, like other benzamide HDACi, [³H]CI-994 exhibits slow binding kinetics when measured in vitro with isolated enzymes and ex vivo when used for autoradiographic mapping of HDAC1–3 density. The regional distribution and density of HDAC1–3 was determined through a series of saturation and kinetics experiments. The binding properties of [³H]CI-994 to HDAC1–3 were characterized and the data were used to determine the regional B_max of the target proteins. K_d values, determined from slice autoradiography, were between 9.17 and 15.6 nM. The HDAC1–3 density (B_max), averaged over whole brain sections, was of 12.9 picomol · mg⁻¹ protein. The highest HDAC1–3 density was found in the cerebellum, followed by hippocampus and cortex. Moderate to low receptor density was found in striatum, hypothalamus and thalamus. These data were correlated with semi-quantitative measures of each HDAC isoform using western blot analysis and it was determined that autoradiographic images most likely represent the sum of HDAC1, HDAC2, and HDAC3 protein density. In competition experiments, [³H]CI-994 binding can be dose-dependently blocked with other HDAC inhibitors, including suberoylanilide hydroxamic acid (SAHA). In summary, we have developed the first known autoradiography tool for imaging class I HDAC enzymes. Although validated in the CNS, [³H]CI-994 will be applicable and beneficial to other target tissues and can be used to evaluate HDAC inhibition in tissues for novel therapies being developed. [³H]CI-994 is now an enabling imaging tool to study the relationship between diseases and epigenetic regulation.     

Solubilization and affinity labeling of a dihydropyridine binding site from skeletal muscle: effects of temperature and diltiazem on [3H]dihydropyridine binding to transverse tubules

Effects of temperature and d-cis-diltiazem (DTZ) on [3H]nitrendipine (NTD) and [3H]nimodipine (NIM) binding to skeletal muscle t-tubular membranes were studied. A decrease in temperature from 37 degrees C to 10 degrees C decreased KD and increased Bmax slightly. DTZ increased binding by increasing Bmax under all conditions and also decreased KD for NTD at 37 degrees C. The binding protein labeled with [3H]isothiocyanate dihydropyridine revealed a molecular weight of 36,000. The binding site for NTD was solubilized by deoxycholate and dihydropyridine binding was still stimulated by DTZ in the solubilized form.