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A fluorescent analogue of propranolol does not label beta adrenoceptor sites

A fluorescent analogue of propranolol does not label beta adrenoceptor sites
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  Brain Research, 181 (1980) 209-213 209 © Elsevier/North-Holland Biomedical Press fluorescent analogue of propranolol does not label beta adrenoceptor sites PETER BARNES, HANNO KOPPEL, PAUL LEWIS, CHRISTINE HUTSON, IAN BLAIR and COLIN DOLLERY Departments of Clinical Pharmacology and H.K. and P.L.) Histopathology, and C.H.) Institute of Child Health, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS U.K.) (Accepted September 6th, 1979) Key words: 9-aminoacridine -- propranolol -- beta-adrenoceptors Recently two fluorescent analogues of propranolol with a high affinity for beta (fl) adrenoceptors have been synthesized 1. Both show specific receptor binding in an isolated adrenaline-dependent adenylate cyclase system. It has been claimed that it is possible by means of fluorescent microscopy to visualize fl-adrenoceptor sites both in the central nervous system 3-5,s and peripheral tissue such as heart 9 and kidneyL Following the report of Hess 6, which casts doubt on this claim, we have re-evaluated one of these compounds, DL-9-amino-acridyl-propranolol (9-AAP) (Yissum Research Development, Hebrew University, Jerusalem). 9-AAP in 0.02 M sodium phosphate buffer, pH 7.4 was injected slowly into the tail vein of 250-300 g male Wistar rats. After 30 min animals were killed by decapitation and the cerebellum was quickly removed and frozen in isopentane cooled to the temperature of liquid nitrogen. After mounting in 'Tissue-Tek II OCT compound' (Raymond A. Lamb, London), sections 12-16 m thick were cut on a cryostat at --22 °C. The frozen sections were mounted on glass slides, dried in air for 20 min, covered with immersion oil and examined under phase contrast and ultraviolet light using a Leitz Orthoplan microscope with an excitator filter BG 12 and absorption filter K430. This technique follows as closely as possible that described by Atlas et al.Z-5,s, 9. Sections from uninjected animals and from animals injected with L- propranolol 5 mg/kg 15 min prior to the injection of 9-AAP were similarly examined. Four animals were studied in each group. Discrete yellow fluorescent dots against a greenish background were visible outlining Purkinje cell perikarya and primary dendrites, using a × 100 objective lens (Fig. 1). This pattern corresponds to that described by Atlas et al. 4. However, fluorescence of the same intensity, distribution and density was also seen in 4 uninjected animals (Fig. 2) and in animals pretreated with L-propranolol. This suggests that the fluorescent particles claimed by Atlas and co-workers to be /3- adrenoceptor sites may be no more than autofluorescent granules. A possible explanation for this discrepancy between our results and those of Atlas et al. could be that the commercially available 9-AAP (claimed by the manufac-  210 Fig. 1. two Purkinje cells in cerebellum of adult rat injected with 9-AAP 5 mg/kg. Unstained, phase contrast (~ 5000). b: as (a), under ultraviolet illumination (~: 5000). turers to have 95 ~ purity) is either impure or inactive. The purity of the commercial compound was assessed using thin layer chromatography with silica GFe54 plates and a solvent system of butanol:acetic acid:water in the proportions 4: 1:4. The compound proved to be inhomogeneous and the major component (Rf = 0.47) comprised approximately 30 % of the total. The major fluorescent band was purified using preparative layer chromatography with the same solvent system and provided the desired compound with more than 95 ~ purity. This purified product was then used in an in vitro binding assay and also injected into a further animal. To determine whether the purified 9-AAP had fl-antagonist activity, a radio- ligand binding assay was employed. Using a preparation of guinea-pig lung membra- nes, binding of the potent fl-adrenoceptor antagonist L[3H]dihydroalprenolol (Radio- chemical centre, Amersham, U.K.) was measured. Specific binding, which represents the amount of ligand bound to fl-adrenoceptor sites, was determined by subtracting the radioactivity bound nonspecifically in the presence of 10 M oL-propranolol from  211 the total radioactivity bound. Displacement of the specific binding by DL-propra- nolol and by 9-AAP was measured and the molar concentration at which there was 50 700 inhibition of specific binding (IC50) calculated. IC50 values of 4 x 10 -s M for DL- propranolol and 5 × 10 -7 M for 9-AAP gave K~ values of 3.2 × 10 -s M and 3.6 × 10 -7 M respectively. This indicates that 9-AAP has/ adrenoceptor antagonist action which is approximately one tenth of the potency of DL-propranolol, confirming the measurements of Atlas and her colleagues 1. These experiments suggest that although the purified 9-AAP appears to be relatively potent as a fl-adrenoceptor antagonist, there is no evidence that it allows specific visualization of fl-adrenoceptor sites. There was no difference in the pattern of fluorescence between untreated rats and one animal injected with the purified 9-AAP in a dose of 5 mg/kg. The finding of Atlas et al. that the pattern of fluorescence is significantly diminished by the prior injection of L-propranolol but not D-propranolol could not be confirmed: in our study there was no change in the pattern or intensity of fluorescence. As these fluorescent dots have been detected in other sites known to have 3-     213 adlenoceptors, it seemed possible that they may be related to presynaptic catechola- mine stores. However, similar fluorescence was also seen in bronchial epithelial cells but not bronchial smooth muscle which makes this explanation unlikely. In the case of the cerebellum, there is good evidence that the structural correlate of the observed autofluorescence may be lipofuscin. We have not been able to detect any fluorescence in the cerebella of young (4, 7, 11 and 21-day-old) rats, which suggests that it is, like lipofuscin, an age-related phenomenon. Purkinje cell perikarya are known to be particularly rich in lysosomes10, and in nerve cells these increase in size and complexity as lipofuscin accumulates in them ll. The lipofuscin inclusions of Purkinje cells have been described as having a strongly yellowish fluorescence7 and the distribution of lipofuscin-positive staining material in Purkinje cells and other neurones7312 appears very similar to the distribution of the pattern of autofluorescence we have demon- strated. In conclusion, commercially available 9-AAP is a mixture of compounds, one of which is a potent p-adrenoceptor antagonist in vitro but we have been unable to determine selective labelling on , adrenoceptor sites in vivo. It is possible that although the fluorescent j3-adrenergic ligand may bind specifically to receptors, the fluorescence would be of too low an intensity to be detected in practice by fluorescence microscopy. PB is supported by MRC Program Grant G971/16/C. 1 Atlas, D. and Levitzki, A., Probing of beta adrenergic receptors by novel fluorescent beta adren- ergic blockers, Proc. nat. Acud. Sci. (Wash.), 14 (1977) 5290-5294. 2 Atlas, D., Melamed, E. and Lahav, M., Beta adrenergic receptors in rat kidney, Lab. Invest., 36 (1977) 465-468. 3 Atlas, D. and Segal, M., Simultaneous visualization of noradrenergic fibers and beta adrenocep- tars in pre- and postsynaptic regions in rat brain, Brain Research, t35 @ ‘7~50. 4 Atlas, D., Teichberg, V. 1. and Changeux, J. P., Direct evidence of beta adrenoceptors on the Pur- kinje cells of mouse cerebellum, Brain Research, 128 (1977) 532-536. 5 Atlas, D. and Melamed, E., Direct mapping of beta adrenergic receptors in the rat central nervous system by a novel fluorescent beta blocker, Brain Research, 150 (1978) 377-385. 6 Hess, A., Visualization of beta adrenergic receptor sites with fluorescent beta adrenergic blocker probes, Brain Research, 160 (1979) 533-538. 7 Issidorides, M. and Shanklin, W. M., Histochemical actions of cellular inclusions in the human neurone, J. Anar. (Lond.), 95 (1961) 151-159. 8 Melamed, E., Lahav, M. and Atlas, D., Histochemical evidence for beta adrenergic receptors in the rat spinal cord, Brain Research, 11 (1976) 511-515. 9 Melamed, E., Lahav, M. and Atlas, D., Beta adrenergic receptors in rat myocardium, Experientia (Basel), 32 (1976) 1387-1389. 10 Palay, S. L. and Chan-Palay, V., Cerebellar Correx, Springx-Verlag, Berlin, 1974. 11 Samorajski, T., Ordy, J. M. and Keefe, J. R., The fine structure of lipofuscin age pigment in the nervous system of aged mice, J. Cell Biol., 26 (1965) 779-795. 12 Sulkin, N. M., The distribution of mucopolysaccharides on the cytoplasm of vertebrate nerve cells, J. Neurochem., 5 (1960) 231-235.
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