The research presented In this thesis provides an extensive study on the extraneuronal uptake of catecholamines in an identified cell type. The extraneuronal uptake of adrenaline, noradrenaline and isoprenaline into smooth muscle cells of guinea-pig trachea!is muscle has been investigated using fluorescence microphotometry to measure catecholamine accumulation in these cells. The principal aim of the study was to obtain information on the transport mechanism responsible for the uptake of catecholamines into smooth muscle cells. However, if fluorescence microphotometry was to be used to measure accumulated catecholamines in cells, it was critical to establish that fluorescence intensity values, although expressed in arbitrary units (F), were linearly related to the amount of amine in the area of tissue measured with the microphotometer. Thus, before commencing the main investigation, this linearity was established in protein model experiments, since it could not be determined directly in tissues. A linear relationship was demonstrated up to values of 3000 F, and this was assumed to apply to measurements made in cells in tissues.
Experiments on guinea-pig trachea were carried out in tissues from animals in which the adrenergic nerves had been destroyed by 6-hydroxydopamine. Rings of tracheae were incubated at 37°C for a specified period in Krebs solution containing no amine (controls) or a concentration of adrenaline, noradrenaline or isoprenaline. The tissues were then washed in amine-free Krebs solution at 0-1°C to remove amine which was in the extracellular space or loosely bound to the tissue. Experiments carried out in the present study showed that a 30 min period for this post-incubation wash was satisfactory. The tissues were then freeze-dried, exposed to formaldehyde gas at 80°C for 1 hr (noradrenaline) or 3 hr (adrenaline or isoprenaline) and embedded in paraffin. Fluorescence intensity measurements in 2.5 urn square areas were made in smooth muscle cells in tissue sections of 7 μm thickness using a Leitz MPV microphotometer.
Analysis of fluorescence intensity values obtained in trachealis smooth muscle cells in different tissues, different animals and different experiments showed that reproducible results were obtained for any particular treatment. This was provided that the experiments were carefully designed and the experimental conditions were rigidly controlled. In addition, it was shown that the fluorescence technique was more sensitive for detecting extraneuronal catecholamines than had been suggested by other workers. Reproducible measurements could be obtained in the smooth muscle cells after incubation of tissues for 5 min in concentrations of adrenaline, noradrenaline or isoprenaline as low as 2 μM.
It was important to prevent loss of catecholamine by intracellular metabolism, subsequent to uptake, if fluorescence measurements were to represent the total amount of amine which entered the cells. A comparative study was carried out with three COMT inhibitors, tropolone, β-thujaplicin and U-0521, which were added to the Krebs solution used in the experiments. The effects of various concentrations of these inhibitors on accumulation of isoprenaline in the smooth muscle cells were examined. Maximum enhancement of isoprenaline fluorescence occurred with 100 μM U-0521, which was used in all subsequent experiments to inhibit COMT. These experiments also showed that COMT in the smooth muscle cells appeared to be fully saturated in tissues incubated in 50 μM and 800 μM, but not 5 μM, isoprenaline. Experiments were also carried out to compare the effects of 100 μM and 300 μM pargyline, an irreversible MAO inhibitor, on the accumulation of noradrenaline in the smooth muscle cells. From these results, a concentration of 100 μM of pargyline was selected to inhibit MAO in all subsequent experiments with noradrenaline and adrenaline. Since isoprenaline fluorescence was unaffected by pargyline, the MAO inhibitor was not included in experiments with isoprenaline. Indirect evidence from the experiments with noradrenaline suggested that MAO in the cells might not be fully saturated even with 800 μM noradrenaline.
Initial rate kinetic experiments on the uptake of adrenaline, noradrenaline and isoprenaline were carried out to establish the kinetic parameters for uptake of the amines into the smooth muscle cells. Tissues were incubated in amine concentrations ranging from 2 μM to 800 μM for 5 min. It had been demonstrated that amine uptake was still occurring at its initial rate after an incubation time of 5 min. The initial rates of uptake obeyed Michaelis-Menten saturation kinetics, showing that the amines were transported into the cells by a carrier-mediated process. The Km and Vmax values obtained by Marquardt's method of non-linear regression analysis were 149 μM and 201 F min-1 for adrenaline, 243 μM and 313 F min-1 for noradrenaline and 266 μM and 256 F min-1 for isoprenaline, respectively. Subsequent results from a total of 25 guinea-pigs gave a mean Km value for adrenaline of 157 μM. From the Km values, it could be concluded that the order of affinities of the three amines for extraneuronal uptake in the guinea-pig trachealis smooth muscle cells was adrenaline > noradrenaline = isoprenaline.
The mechanisms of action of three inhibitors of extraneuronal uptake, phenoxybenzamine, normetanephrine and corticosterone, were also determined from initial rate kinetic experiments. Initial rates of uptake of adrenaline were determined in tissues incubated in concentrations of the amine ranging from 50 μM to 300 μM, in the absence or presence of 10 μM of the inhibitor. The experiments confirmed that phenoxybenzamine was an irreversible inhibitor of extraneuronal uptake in the smooth muscle cells. Normetanephrine and corticosterone were shown to be reversible, competitive uptake inhibitors. Kinetic analysis of the data for these two reversible inhibitors was used to obtain K𝑖 values: 2.8 μM for normetanephrine and 1.5 μM for corticosterone. These values showed that both inhibitors had high affinities for the extraneuronal uptake mechanism in the smooth muscle cells.
Further experiments were carried out to establish whether the carrier-mediated uptake of catecholamines into the cells occurred by an active transport or by facilitated diffusion. It was shown that the uptake of adrenaline was reduced by anoxia plus glucose deprivation, by ouabain and by decreasing the sodium ion concentration or increasing the potassium ion concentration. From these results, it was concluded that uptake of adrenaline into the smooth muscle cells occurred by a secondary active transport linked to Na+/K+-dependent ATPase.
This study has provided quantitative results on extraneuronal uptake,using fluorescence microphotometry. These results are complementary with those obtained by other workers using radiolabelled catecholamines in extraneuronal uptake studies. The major advantage of the microphotometric technique is that it provides measurements of the uptake into cells which are identified under the microscope, rather than measurements of uptake relating to a heterogeneous group of cell types in a tissue. The kinetic studies in guinea-pig trachealis smooth muscle cells showed that the affinities of adrenaline, noradrenaline and isoprenaline for extraneuronal uptake were of the same order of magnitude. However, it was of interest that adrenaline had about a two-fold higher affinity than the other two amines for the uptake mechanism. This is contrary to the accepted generalization, based on the results of earlier studies in rat heart, that the order of substrate affinities for extraneuronal uptake was isoprenaline > adrenaline > noradrenaline. It was also concluded that "extraneuronal O-methylation" behaved as a relatively high affinity system in the smooth muscle cells, in contrast with the low affinity of the actual uptake mechanism and of "extraneuronal deamination". This conclusion was reached by considering the relationship between the Km values of the amines for extraneuronal uptake and the saturability of COMT and MAO, which was deduced indirectly from the results of experiments with COMT and MAO inhibitors. The extraneuronal uptake inhibitors, normetanephrine and corticosterone, both had much higher affinities for extraneuronal uptake in the trachealis smooth muscle cells than the substrate adrenaline. The possible physiological significance of this finding has been considered.
In conclusion, this study has demonstrated that fluorescence microphotometry is a useful technique for obtaining information on extraneuronal transport mechanisms in specific cells. If its use can be extended to similar cell types and to different cell types in various species, it may aid in resolving the question of whether extraneuronal uptake mechanisms in different cell types and species show some homogeneity or are heterogeneous.