A histochemical study of the adrenergic innervation and of extraneuronal accumulation of noradrenaline in the guinea-pig lung

Saar, Nili (1979). A histochemical study of the adrenergic innervation and of extraneuronal accumulation of noradrenaline in the guinea-pig lung PhD Thesis, School of Biomedical Sciences, The University of Queensland. doi:10.14264/uql.2014.464

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Author Saar, Nili
Thesis Title A histochemical study of the adrenergic innervation and of extraneuronal accumulation of noradrenaline in the guinea-pig lung
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
DOI 10.14264/uql.2014.464
Publication date 1979
Thesis type PhD Thesis
Supervisor S. R. O'Donnell
Total pages 158
Language eng
Subjects 0601 Biochemistry and Cell Biology
Formatted abstract
Fluorescence histochemistry was used to study the extraneuronal accumulation of NA in the guinea-pig lung. In order to specifically identify fluorescence of exogenous NA in non-neuronal tissue, without interference from the fluorescence of endogenous NA in adrenergic nerves, a study of the adrenergic innervation of the guinea-pig lung was also carried out.

The guinea-pig lung contained a sparse adrenergic innervation located in airway smooth muscle and in the vasculature. The density of adrenergic nerves decreased from the laryngeal end of the trachea, which was fairly densely innervated, to the bronchioles, which contained only occasional adrenergic fibres. Adrenergic innervation to pulmonary and bronchial blood vessels was more conspicuous than that to the airway smooth muscle and was prominent in the small pulmonary and bronchial arterioles. The relatively sparse innervation seen in the airways and the denser innervation to pulmonary vasculature, particularly small arterioles, suggested that adrenergic mechanisms are important at the pulmonary and bronchial arteriolar level. The sparse adrenergic innervation to air\\7ays suggests that inhibitory (bronchodilation) control, particularly of smaller airways, either involves limited direct sympathetic innervation coupled with myogenic propagation or may involve non-adrenergic, non-cholinergic nerves.

Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) or guanethidine was attempted so that extraneuronal accumulation of NA in lung could be studied without interference from the fluorescence of endogenous NA or from loss of NA into adrenergic nerves. Chemical sympathectomy was achieved in trachea and lung after a single dose of 50 mg/kg (i.v. or i.p.) of 6-OHDA. Sympathectomy was shown directly by the lack of fluorescent nerve terminals in trachea and lung from treated animals and the failure of α-methyl noradrenaline (α-Me NA) to restore fluorescence.

Chemical sympathectomy in trachea was confirmed by (a) decreased uptake and retention of radioactivity following incubation of the trachea in 3H-NA (5 x 10-8M) and (b) supersensitivity to NA of isolated tracheal chain preparations from 6-OHDA treated animal. Treatment of new-born guinea-pigs with 6-OHDA (50 mg/kg daily s.c. for 7 days) or treatment of adult guinea-pigs with guanethidine (25 or 30 mg/kg daily i.p. for 6 weeks) did not produce destruction of adrenergic nerves in peripheral tissues studied.

Extraneuronal accumulation of NA was visualized in lungs which had been either perfused with NA or obtained from animals exposed to NA aerosol. A number of cellular sites in the lung accumulated NA, namely vascular and airway smooth muscle, epithelial lining of the airways, fibroblasts, cartilage cells and septal cells in respiratory tissue. Accumulation of NA in vascular pulmonary smooth muscle was evident only in lungs perfused with NA and not in lungs from animals' exposed to NA aerosol. This suggested that, following NA aerosol to conscious animals, NA was taken up in other sites e.g. cells in the airways and in respiratory tissues thus preventing both the accumulation in vascular smooth muscle and the entry of NA into the systemic circulation.

The accumulation of NA in tracheal smooth muscle was further characterized using quantitative fluorescence microscopy. The accumulation process of NA in tracheal smooth muscle was saturable with an apparent Km value of 1.56 x 10-4M, comparable with that of other extraneuronal uptake processes. The accumulation of α-Me NA in tracheal smooth muscle was increased by inhibition of COMT with 2 x 10-4M of tropolone or β-thujaplicin but not by the same concentration of 3,4-dimethoxy-5-hydroxybenzoic acid. The accumulation of NA was increased after inhibition of MAO by pretreatment of animals with nialamide. Thus NA accumulation in tracheal smooth muscle is influenced by both MAO and COMT activity. The accumulation in lung was also influenced by the activity of MAO and COMT. Inhibition of MAO and COMT in the perfused lung and of MAO in the animal receiving NA aerosol markedly enhanced fluorescence. Histochemical staining for MAO activity confirmed the presence of MAO activity in lung and was located in sites which accumulated NA.

The accumulation in airway and vascular smooth muscle, fibroblasts, cartilage cells and in bronchial epithelium was inhibited by the extraneuronal inhibitor drugs, phenoxybenzamine, metanephrine and methoxyisoprenaline. This accumulation was also prevented if the perfusion with NA was carried out at 0-2°C rather than at 37°C. This accumulation thus displayed similar properties to the extraneuronal accumulation of NA described in smooth muscle which is thought to be mediated by a carrier transport system and to have a low affinity for NA.

The accumulation of NA in septal cells of respiratory tissue was prevented by perfusion with NA at reduced (0-2°C) temperature but not by the extraneuronal inhibitor drugs. This accumulation probably corresponds to the accumulation process of NA in pulmonary vascular endothelia in lungs of other species which is thought to represent an uptake process with high affinity for NA.

The marked increase in NA accumulation in lung following inhibition of the metabolizing enzymes in contrast to the lack of neuronal uptake inhibition (by sympathectomy) on NA accumulation suggested that the extraneuronal accumulation and metabolism rather than neuronal uptake is the major process for the removal and inactivation of NA in guinea-pig lung, particularly when the lung is exposed to high concentrations of NA.
Keyword Noradrenaline
Guinea pigs -- Physiology
Adrenergic mechanisms
Additional Notes Other Title: A histochemical study of the adrenergic innervation
Other Title: Extra neuronal accumulation in guinea-pig lungs.

Document type: Thesis
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