Autoradiographic mapping of the glucose transporter with cytochalasin B in the mammalian eye.

The anatomic localization of the glucose transport protein in the eyes of rats, rabbits, baboons, marmosets, and humans with [3H]cytochalasin B using in vitro autoradiography showed high densities of glucose carrier densities. These densities were seen in the ciliary body, especially ciliary processes, iris, retina, and in some species, the trabecular meshwork and lens. In the lens, specific [3H]cytochalasin B-binding sites were mainly concentrated in the lens nucleus. Lower concentrations were found in the cortex. During aging, glucose transporter concentration increases up to the age of 8 yr in the marmoset lens nucleus, but decreases in the cortex and retina. Moderate amounts of carrier are located in the corneal endothelium and epithelium. The enrichment of glucose carrier protein in the trabecular meshwork suggests a high metabolic activity and a possible relationship in the regulation of intraocular pressure.

Quantitative autoradiography of [3H]norharman [( 3H]beta-carboline) binding sites in the rat brain.

The anatomical distribution of [3H]norharman binding sites was determined by quantitative autoradiography in rat brain slices. They are enriched in hypothalamic, thalamic, accumbens and amygdaloid nuclei as well as in hippocampal, neocortical and olfactory-related structures. The distribution pattern differs from that of other previously described receptors or binding sites (e.g. monoamine oxidase, benzodiazepine, tryptamine, 5-hydroxytryptamine receptors (5-HT1A, 5-HT1B, 5-HT1C, 5HT2], which suggests that a unique class of [3H]norharman binding sites exists in the rat brain. The findings are consistent with previous experiments which showed high affinity binding sites for [3H]norharman in rat brain membranes (KD 1.552 nM; autoradiography KD 5.5 nM). A correspondence in the displacing activity of drugs was found for both methods (crude membrane fraction: harman much greater than tryptamine much greater than 5-hydroxytryptamine greater than N-methyl-beta-carboline-3-carboxamide (FG 7142) = diazepam; autoradiography: harman much greater than tryptamine much greater than FG 7142 greater than 5-hydroxytryptamine greater than diazepam). Provided that the binding sites represent functional receptors, the present anatomical findings may explain the biological effects of norharman, e. g. pro-conflict behaviour (limbic-hypothalamic structures), tonic-clonic convulsions (limbic-cortical structures) and alterations of locomotor activity (accumbens nucleus).

Autoradiographic analysis of benzodiazepine receptors in mutant mice with cerebellar defects.

[3H]Flunitrazepam binding sites were analysed in the cerebellar cortex, deep cerebellar and vestibular nuclei of Purkinje cell deficit (pcd) mutant mice which have an almost complete postnatal loss of Purkinje cells, and weaver mutant mice, in which there is a postnatal degeneration of granule cells. Increases in [3H]flunitrazepam binding site densities were found in the molecular and granule cell layer of weaver mutant mice, whereas decreases were observed in pcd mutant mice when compared to wildtype 'control' mice. Apparently unaltered benzodiazepine receptor densities were found in the flocculus of pcd mutant mice, whereas increases were seen in the flocculus of weaver mutant mice. The densities of benzodiazepine binding sites in the medial and lateral deep cerebellar nuclei of both mutants significantly exceeded those in control mice. Significant increases in flunitrazepam binding sites were also found in the superior and spinal nucleus of the vestibular complex of pcd mice as compared to wildtype. In the weaver mutants the benzodiazepine receptor density is enhanced in the superior and medial vestibular nucleus. Apparently unaltered numbers of such receptors compared to the wildtype control group were found in the remaining vestibular nuclei of both mutants.

Quantitative autoradiographic localization of [125I]alpha-bungarotoxin binding sites in the honeybee brain.

Binding sites for the nicotinic acetylcholine receptor antagonist, [125I]alpha-bungarotoxin, were localized in the honeybee brain by in vitro autoradiography. Highest binding site densities were localized in the suboesophageal ganglion, the optic tubercles, optic lobes medulla and lobula, antennal lobes, dorsal lobes and the alpha-lobes of the mushroom bodies. The distribution pattern of these putative nicotinic acetylcholine receptors suggests that acetylcholine is involved in several sensory pathways and in central information processing in the honeybee brain.

Characterization and quantitative autoradiography of [3H]tryptamine binding sites in rat brain.

[3H]Tryptamine binds with high affinity (Kd = 9.1 nM, Bmax = 54 fmol/mg wet wt.) to tissue sections of rat brain. The binding occurs rapidly and is reversible. Low concentrations of the beta-carbolines harmaline (IC50 = 25 nM) and tetrahydronorharman (tetrahydro-beta-carboline, IC50 = 50 nM) inhibit [3H]tryptamine binding. Serotonin (5-HT, IC50 = 2600 nM) as well as the 5-HT receptor antagonists methysergide and metergoline displace [3H]tryptamine at much higher concentrations from brain slices. The distribution of [3H]tryptamine binding sites in sections of rat brain has been analyzed by quantitative autoradiography. The highest density of binding sites is found in the nucleus (n.) interpeduncularis, a slightly lower one in the locus coeruleus. Moderately labelled are the n. accumbens septi, n. septi lateralis, n. medialis habenulae, n. tractus olfactorii lateralis, the central region of the amygdala, n. caudatus/putamen, n. reuniens and the hippocampal formation. A low density of binding sites is detected in the cerebral cortex and the subiculum. Even less binding sites are found in the n. dorsalis raphe and the substantia nigra. The pattern of distribution of [3H]tryptamine binding sites differs from that of [3H]5-HT (5-HT1), [3H]ketanserin (5-HT2) as well as [3H]imipramine binding sites. These data suggest unique tryptamine binding sites.

Autoradiographic mapping of a selective cyclic adenosine monophosphate phosphodiesterase in rat brain with the antidepressant [3H]rolipram.

Rolipram is a clinically effective antidepressant with selective cAMP phosphodiesterase (PDE) inhibiting properties. (+/-)-[3H]Rolipram binds with high affinity (Kd = 2.52 +/- 0.47 nM) to sections of rat brain (Hill number = 0.90 +/- 0.05). Binding is stereospecific. Association of (+/-) [3H]rolipram to sections is rapid (47% of specific binding in the first minute, kobs = 0.52 min-1). Dissociation of (+/-)-[3H]rolipram exhibits non first order kinetics (3 component model; t1/2 = 2.5 min, 50 min and 6 h, respectively). A number of PDE inhibitors reduce (+/-)-[3H]rolipram binding to the level of nonspecific binding ((-)-rolipram, IC50 = 0.9 nM; (+/-)-rolipram, IC50 = 1.5 nM; Ro 20-1724, IC50 = 11 nM; ICI 63.197, IC50 = 35 nM; medazepam, IC50 = 240 nM; diazepam, IC50 = 1200 nM; IBMX, IC50 = 3800 nM). In vitro autoradiography reveals high binding site densities in the cerebellum, olfactory bulb, lateral septal nucleus, frontal cortex, subiculum and CA1 of hippocampus. Most of the labeled structures are part of the limbic system. In vivo autoradiography of (+/-)-[3H]rolipram binding shows much more nonspecific binding than in vitro, nevertheless the distribution pattern of (+/-)-[3H]rolipram binding sites is similar. A comparison of the distribution pattern of (+/-)-[3H]rolipram binding sites with that of an antidepressant (monoamine oxidase inhibitor, monoamine uptake inhibitor) reveals no overlap. Limited, though significant correlations exist with the distribution of beta 1-adrenergic, adenosine1 and glutamate/quisqualate receptors as well as protein kinase C, but not with beta 2-adrenergic receptors and forskolin binding sites.

Quantitative autoradiographic localization of alpha 2-antagonist binding sites in rat brain using [3H]idazoxan.

The distribution of alpha 2-receptors was determined by quantitative autoradiography with the antagonist [3H]idazoxan. Apart from regions which are known to be enriched in alpha 2-receptors, the highest silver grain densities were located over the subfornical organ and the area postrema. However, binding in these regions was not displaced by yohimbine and therefore, according to the present understanding, cannot be considered to represent alpha 2-receptors. Within the cerebellum, alpha 2-receptors were found to be arranged in 3 sagitally oriented strips within the molecular layer of lobules 9 and 10, suggesting a co-incidence with dopamine and substance P receptors in this structure.

Autoradiographic localization of [3H]buspirone binding sites in rat brain.

Binding sites for [3H]buspirone were identified on sections from rat brain by light microscopic autoradiography. Incubated sections were processed by the Ultrofilm as well as the coverslip method. Highest densities of [3H]buspirone binding were localized in the caudate/putamen, nucleus accumbens septi, olfactory tubercle and in the glomerula of the olfactory bulb. This distribution pattern is different from any serotonin receptor but fits well with that of dopamine receptors. Therefore, this study supports the hypothesis that buspirone acts, at least partially, on dopamine receptors.

Adaptive changes of dopamine-D2 receptors in rat brain following ethanol withdrawal: a quantitative autoradiographic investigation.

The effect of subchronic treatment with two doses of ethanol (5 and 10 vol% drinking fluid) on the density of dopamine-D2 receptors was investigated at two different phases of withdrawal, namely 24 h and 5 days after the cessation of the ethanol application. The number of dopamine-D2 receptors was affected in regions receiving projections from both the substantia nigra as well as the ventral tegmentum. Twenty-four hours after the replacement of the ethanol solution by water, a dose-dependent decrease of D2 receptors was found in all regions (N. caudatus dorsalis, medialis and ventralis, N. accumbens lateralis and medialis, tuberculum olfactorium) and most of the analyzed planes [interaural 7.7-10.2 according to the atlas of Paxinos and Watson (35)]. At day 5 of withdrawal, the number of dopamine-D2 receptors of the animals treated with 5 vol% ethanol reached the level of water controls in most planes. In contrast, two- to three-fold higher numbers were detected in animals treated with the higher dose. Only in the most caudal parts of the investigated regions, was the number of receptors decreased with the higher dose. The mesocorticolimbic system seems to be less sensitive to the effects of ethanol than the nigrostriatal neurones. The findings of the present study suggest an increased activity of dopaminergic neurons with an adaptive reduction of dopamine-D2 receptors during the subchronic treatment with ethanol during the first day(s) of withdrawal. This phase is followed by a reduced turnover rate for up to 7 days (21). The reduced activity of dopaminergic neurones induces a compensatory increase of the number of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Increase in beta-adrenergic receptors in rabbits in long-term local administration of beta-blockers]. / Zunahme von beta-adrenergen Rezeptoren bei Kaninchen unter lokaler Langzeitapplikation von beta-Blockern.

Chronic application of beta-blockers often induces tachyphylaxia of unknown origin. After long-term topical pretreatment of pindolol and timolol in rabbit eyes (up to 12 weeks, twice a day) beta 2-adrenergic receptors were localized and quantified with autoradiographic methods. Frozen sections of albino rabbit eyes were labelled with [125I] cyanopindolol. Quantification revealed a significant increase in the density of beta 2-receptors after premedication with pindolol in the ciliary body and the corneal epithelium. This increase was detectable after 2 weeks of premedication and reached its maximum after 4 weeks. After premedication with timolol a significant increase of beta 2-receptors in the epithelium of the ciliary body and the corneal epithelium was visible. Following pindolol over an equivalent time-course, to that of timolol administrations, an increasing number of beta 2-receptor sites was also observed. No significant changes were visible for either kind of premedication investigated (pindolol/timolol) in the corneal endothelium, the lens or the choroid. Chronic application of topically instilled beta-blocking agents leads to a significant increase in receptor density in ocular structures that are involved in aqueous humor production. This up-regulation of beta 2-adrenergic receptors might explain the intraocular events at receptor level in relation to the phenomenon of tachyphylaxis.

[Localization of cAMP-selective phosphodiesterase in the mammalian eye by (3H)-rolipram]. / Lokalisation von cAMP-selektiver Phosphodiesterase am Säugetierauge durch [3H]-Rolipram.

It has been shown that inhibition of phosphodiesterase (PDE) or an increase in intracellular cyclic adenosine monophosphate (cAMP) can lower the intraocular pressure in mammalian eyes. The PDE inhibitor rolipram binds with high affinity to a cAMP-dependent PDE. In the following study, receptors for rolipram in the mammalian eye are determined by autoradiography. About 1050 histological sections of rabbit (albino and pigmented), rat, monkey (baboon and marmoset) and human eyes were examined concerning rolipram-binding sites in different structures. The highest specific binding of rolipram to the ciliary body epithelium was seen in human, rabbit and baboon eyes. Marmoset eyes showed the highest specific binding in the ciliary body muscle, rat eyes in the retina. Specific binding was consistently high in the ciliary body epithelium and therefore at the site of aqueous humor formation. Because of these results and the known mode of action of rolipram, one can assume a close connection between rolipram-binding sites and the regulation of intraocular pressure.

Quantitative autoradiography of beta-adrenergic receptors in rabbit eyes.

Beta-adrenergic receptors were localized and quantified with autoradiographic methods in frozen sections of eyes of albino rabbits using [125I]cyanopindolol and [3H]dihydroalprenolol. Quantification in albino rabbits revealed highest beta 2-adrenoceptor densities in the ciliary body, the corneal epithelium and the corneal endothelium. Moreover, the iris showed significant amounts of specific binding, whereas the retina and sclera are nearly free of these receptors.

[Quantitative autoradiography with (125I)cyanopindolol of ocular tissues]. / Quantitative Autoradiographie mit [125I]Cyanopindolol an okulären Geweben.

In the present study beta-adrenergic receptors were identified and localized by autoradiographic methods using [125I]cyanopindolol in human eyes, as well as in the eyes of marmosets and albino rabbits. Quantification revealed that the highest binding-site densities are in the ciliary body and the corneal epithelium and endothelium. Moreover, the iris and conjunctiva showed significant amounts of specific [125I]cyanopindolol binding. In pigmented human and marmoset eyes, binding to the ciliary epithelium and iris was not inhibited by 10 microM propranolol, thus representing nonspecific binding sites. In nonpigmented rabbit eyes, binding to all parts of the eye was reduced to the level of background by 10 microM propranolol and 100 nM ICI 118-551, which is a highly selective beta 2-adrenergic ligand. Being selective for serotonin1B receptors, 100 nM ICI 89-406 which blocks more than 95% of the beta 1-adrenergic receptors, and RU 24,969 have no effect on [125I]cyanopindolol binding, which suggests that there is binding to beta 2-adrenergic receptors. The anatomical location and quantification of beta 2-receptors in the mammalian eye may contribute to better understanding of beta 2-adrenoceptor function and the action of beta-adrenoceptor blocking agents in glaucoma therapy.

Quantitative autoradiographic distribution and pharmacological characterization of (3H)buspirone binding to sections from rat, bovine and marmoset brain.

(3H)Buspirone binds with high affinity (KD = 11 nM) to sections from rat striatum. Spiroperidol, chlorpromazine, (+)-butaclamol and apomorphine are the most potent inhibitors of (3H)buspirone binding. Ketanserin, SCH 23390, serotonin and phentolamine are clearly less active. The regional distribution of (3H)buspirone binding in rat and marmoset brain is characterized by high silver grain densities in the olfactory tubercle, nucleus accumbens and striatum. In the hypophysis, the pars intermedia is strongly labeled. Within the hippocampal formation, slightly higher binding site densities are found in the dentate gyrus. The distribution pattern of binding sites in the dentate gyrus varies according to the species investigated. The data presented in this study permit the conclusion that (3H)buspirone binds with high affinity to dopamine 2 receptors but do not exclude additional binding to other types of receptors, e.g. 5-HT1 receptors. The interaction of buspirone with dopamine 2 receptors may be mainly responsible for its pharmacological profile.

Cytomegalovirus retinitis under combination therapy with zidovudine and dideoxycytidine in advanced human immunodeficiency virus infection.

The incidence of cytomegalovirus (CMV) retinitis during combination therapy with zidovudine (azidothymidine AZT) and zalcitabine (dideoxycytidine ddC), was compared with that during monotherapy with AZT alone in patients with advanced human immunodeficiency virus (HIV) infection. A total of 85 patients with CD4 cell counts under 500/microliters were enrolled in a prospective, controlled study. Between August 1991 and June 1992, these patients were treated daily with 500 mg/kg AZT given alone (n = 42) or in combination with 0.02 mg/kg ddC (n = 43). The rate of occurrence of typical microvascular retinopathy with cotton-wool exudates was lower in patients receiving combination treatment than in those given monotherapy [10 patients (26%) receiving AZT/ddC vs 23 patients (56%) given AZT; P < or = 0.01, chi-square test]. CMV retinitis developed in 8 patients (19%) treated with AZT and in 6 patients (14%) treated with ddC and AZT (no significant difference). In contrast to recently published data, we found no decrease in the rate of occurrence of retinitis in the group under combined antiretroviral therapy but observed a significantly lower incidence of microvascular retinopathy.

[Quantitative autoradiography of the glucose transport protein with (3H)cytochalasin-B in the mammalian eye]. / Quantitative Autoradiographie des Glukosetransportproteins mit [3H]Cytochalasin-B im Säugerauge.

In vitro autoradiography with [3H]cytochalasin for the anatomic localization of the glucose transport protein in eyes of rats, rabbits, baboons, marmosets and humans reveals high glucose carrier densities in the ciliary body, especially ciliary processes, iris, retina and, in some species, in the trabecular meshwork and lens. In the lens, specific [3H]cytochalasin B binding sites are mainly concentrated in the lens nucleus. Lower concentrations are found in the cortex. During again, the glucose transporter concentration increases up to the age of 8 years in the marmoset lens nucleus, but decreases in the cortex and retina. Moderate amounts of carrier are located in the corneal endo- and epithelium. These findings suggest a possible involvement of glucose transporter-related mechanisms in cataractogenesis, e.g. in that resulting from glycosylation of lens proteins. The enrichment of glucose carrier protein in the trabecular meshwork suggests a high metabolic activity and a possible involvement in the regulation of intraocular pressure. The relationship between glucose transport and utilization is also briefly discussed.