Demonstration of catecholamine and resorcinolamine derivatives as formaldehyde-induced fluorescence in protein models.

We assessed in protein droplet models the potential use of the formaldehyde condensation method for histochemical demonstration of a wide range of catecholamines and resorcinolamines. The experiments showed that all of the amines tested, except salbutamol and carbuterol, formed fluorophores, and that the fluorescence was specific [i.e., there was no fluorescence in the absence of formaldehyde, the fluorescence was quenched by water, and the fluorophores were subject to photodecomposition by the exciting (405-nm) light]. Peak wavelengths of the emission spectra were 480-485 nm for fluorophores of resorcinolamine derivatives. The fluorescence intensity of the catecholamines was greater than that of the resorcinolamines. Fluorophore formation was not hindered by substitution of t-butyl, phenylisoprophyl, or p-hydroxyphenylisopropyl on the amino-N in catecholamines (t-butylnorepinephrine, Cc24, Cc25, respectively) or resorcinolamines (terbutaline, Th1161, fenoterol, respectively), and fluorophores also formed for catecholamines with the amino-N in a ring structure (rimiterol) or with a long alkyl chain substituted on the amino-N (hexoprenaline). Our study showed that fluorescence microphotometry can be used to detect a range of drugs that are catecholamines or resorcinolamines, and hence it should be possible to use this technique to study the properties of dissipation of these amines in tissues.

Stereoselectivity of extraneuronal uptake of catecholamines in guinea-pig trachealis smooth muscle cells.

The extraneuronal uptake of the (-)- and (+)-isomers of three catecholamines, isoprenaline, adrenaline and noradrenaline, were compared in guinea-pig trachealis smooth muscle cells, by a fluorescence microphotometric method. Preliminary experiments showed that the initial rates of uptake of the (-)-isomers were greater than those of the (+)-isomers in tissues incubated in 25 microM adrenaline or noradrenaline or 50 microM isoprenaline. More detailed experiments showed that the Km values of the (+)-isomers of the amines for extraneuronal uptake were not significantly different from each other, but the Km value of (-)-isoprenaline less than (-)-adrenaline less than (-)-noradrenaline. Thus, the order of the ratios of the Km values for the (+):(-)-isomers was isoprenaline greater than adrenaline greater than noradrenaline. The results showed that there is stereoselectivity of the extraneuronal uptake of catecholamines, but it is greatest for isoprenaline (4.9 fold), less for adrenaline (2.5 fold) and almost negligible (1.6 fold) for noradrenaline.

Dissipation mechanisms for 5-hydroxytryptamine in the coronary circulation of the isolated perfused heart of the rat.

1. The contribution of uptake into vascular endothelial cells, of neuronal uptake and of extraneuronal uptake in the dissipation of 5-hydroxytryptamine (5-HT) perfused through the coronary circulation of the rat heart was examined. 2. Hearts from reserpine-pretreated rats were isolated and perfused in vitro with 5-HT, in the absence or presence of inhibitors, and rates of appearance of the deaminated metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in the venous effluent were measured using an h.p.l.c. assay. 3. The steady-state rates of 5-HIAA appearance in the venous effluent in hearts perfused with 1 microM 5-HT (422 +/- 8.48 pmol g-1 min-1, n = 12) were reduced by pretreatment of the rats with 6-hydroxydopamine (22% inhibition), and by inclusion in the perfusion solution of 30 microM cocaine (28% inhibition), 100 microM 3-O-methylisoprenaline (64% inhibition), 100 microM corticosterone (58% inhibition), or 30 microM cocaine and 100 microM 3-O-methylisoprenaline (87% inhibition). 4. The extraneuronal deamination of 5-HT in the heart was saturable (Km = 101 microM, Vmax = 31.2 nmol g-1 min-1). The neuronal deamination of 5-HT was saturated by about 50 fold lower concentrations of 5-HT than was extraneuronal deamination, but Km and Vmax values could not be determined. 5. In the coronary circulation of the rat heart, 5-HT was dissipated by the uptake processes for catecholamines, extraneuronal uptake (predominantly) and neuronal uptake, and subsequent metabolism by monoamine oxidase. There was no evidence that a cocaine-sensitive uptake of 5-HT into vascular endothelial cells made any significant contribution to 5-HT dissipation in the heart.

The extraneuronal accumulation of isoprenaline in trachea and atria of guinea-pig and cat: a fluorescence histochemical study.

1 The Falck-Hillarp histochemical technique was used to locate extraneuronal sites of accumulation of isoprenaline in trachea and atria from guinea-pig and cat. With a tissue exposure time to formaldehyde gas of 3 h, isoprenaline was located as green fluorescence.2 Quantitative microphotometry was used to measure fluorescence intensity within cells in the trachealis smooth muscle and the atrial myocardium of both species.3 After incubation of tissues in 50 muM isoprenaline, specific fluorescence was seen in trachealis smooth muscle of both species and in the atrial myocardium of cat but not guinea-pig. In both species, fluorescence was also seen in the chondroblasts of the tracheal cartilage and in blood vessels in all tissues.4 In trachealis smooth muscle of both species and in cat atrial myocardium, fluorescence brightness, resulting from incubation of tissues in 50 muM isoprenaline was significantly increased by 200 muM beta-thujaplicin, an inhibitor of catechol-O-methyl transferase (COMT). In the presence of beta-thujaplicin, fluorescence was not visible in guinea-pig atrial myocardium with 50 muM isoprenaline, although fluorescence brightness measured in myocardial cells was now greater than that in corresponding controls.5 The fluorescence intensity seen in cat and guinea-pig trachealis smooth muscle cells and in cat atrial myocardial cells after incubation in 50 muM isoprenaline was decreased significantly in the presence of phenoxybenzamine (100 muM). In guinea-pig atria, phenoxybenzamine had no effect on myocardial fluorescence. Fluorescence intensity was also decreased if the incubation with isoprenaline was carried out at 0 degrees C or if the post-incubation washing temperature was 37 degrees C instead of 0 to 2 degrees C.6 The results demonstrate that the fluorescence histochemical technique can be used to locate isoprenaline in tissues. They also indicate that guinea-pig and cat trachealis smooth muscle cells and cat atrial myocardial cells can accumulate isoprenaline (a) by a mechanism sensitive to phenoxybenzamine and (b) into sites in which COMT plays a functional role in inactivating isoprenaline at the concentration used in these histochemical experiments (50 muM). In contrast, the guinea-pig atrial myocardial cells may have a minimal capacity to accumulate isoprenaline by a phenoxybenzamine-sensitive uptake mechanism.

The effects of thyroxine treatment of rats on neuronal and extraneuronal uptake and metabolism of catecholamines in the heart.

The effects of thyroxine (T4) treatment of rats on the neuronal and extraneuronal uptake and subsequent metabolism of catecholamines in the heart were examined, to determine whether changes in the local dissipation of catecholamines might contribute to the enhanced sympathetic cardiac responses that occur when thyroid hormone levels are elevated. T4-treated rats were injected subcutaneously with L-thyroxine sodium 1 mg kg(-1) on days 1, 3 and 5, and controls were injected with the normal saline vehicle on the same days. The experiments on isolated, perfused hearts were carried out on day 8. The T4-treated rats had only 50% of the growth rate of the controls and their heart weights were 18% greater than the controls. The experimental data were adjusted to allow for the increase in heart weight caused by the T4 treatment. The initial rates of neuronal uptake of noradrenaline and of extraneuronal uptake of noradrenaline and isoprenaline in the hearts from T4-treated rats were not significantly different from those in hearts from control rats. The steady-state rates of extraneuronal O-methylation of isoprenaline and of extraneuronal deamination of noradrenaline in hearts from T4-treated rats were not significantly different from those in hearts from control rats. The steady-state rate of neuronal deamination of noradrenaline was significantly lower and the accumulation of unmetabolized noradrenaline in the hearts was significantly greater in T4-treated rats than in the controls. These findings could be explained by a decrease in neuronal monoamine oxidase activity or by an increase in intraneuronal binding of noradrenaline in hearts from T4-treated rats. 6 The study has shown that it is unlikely that increased plasma thyroid hormone levels cause cardiac supersensitivity to catecholamines by affecting the local dissipation processes for those amines in the heart.

Some factors affecting the extraneuronal accumulation of adrenaline in guinea-pig trachealis smooth muscle cells.

Tracheal segments from guinea-pigs pretreated with 6-hydroxydopamine were incubated in 5 or 200 mumol X 1(-1) adrenaline at 37 degrees C. Catechol-O-methyltransferase and monoamine oxidase were inhibited by U-0521 and pargyline, respectively, and the accumulation of adrenaline in trachealis smooth muscle cells was measured by fluorescence microphotometry. The effects of anoxia, anoxia plus glucose deprivation, ouabain, reduced sodium (Na+) concentration, elevated potassium (K+) concentration and calcium (Ca2+) deprivation on adrenaline accumulation were examined in tissues incubated in adrenaline for 5 min. Adrenaline accumulation in the trachealis smooth muscle cells was reduced by inhibition of oxidative metabolism (anoxia and glucose deprivation), by ouabain (100 mumol X 1(-1), by a reduction in Na+ concentration from 139 mmol X 1(-1) to 25 mmol X 1(-1), and by increases in K+ concentration, but not by omission of Ca2+. These results from guinea-pig trachealis smooth muscle cells, in combination with those obtained by other workers on various organs and tissues, indicate that the extraneuronal accumulation of catecholamines requires energy derived from oxidative metabolism and is very sensitive to inhibition by increases in K+ concentration. However, there is marked variability in the effects of ouabain and of reduced Na+ or Ca2+ concentrations on the accumulation.

Kinetic analyses of the mechanisms of action of inhibitors of the extraneuronal uptake of adrenaline in smooth muscle cells of guinea-pig trachea.

1. Tracheal segments from guinea-pigs pretreated with 6-hydroxydopamine were incubated with adrenaline (50--300 microM) at 37 degrees C for 5 min in the absence or presence of an extraneuronal uptake inhibitor (normetanephrine, corticosterone or phenoxybenzamine). Catechol-O-methyl transferase and monoamine oxidase were inhibited by 100 microM U-0521 and pargyline, respectively. Tissues were prepared for fluorescence histochemistry, and accumulated adrenaline in trachealis smooth muscle cells was measured by fluorescence microphotometry. Initial rates of adrenaline uptake were calculated. 2. In the absence of an extraneuronal uptake inhibitor, adrenaline uptake obeyed Michaelis-Menten saturation kinetics. The mean Km value for adrenaline from a total of 25 guinea-pigs was 157 microM. 3. The mechanisms of action of three extraneuronal uptake inhibitors were determined by analysis of initial rate kinetic data for adrenaline uptake. The results showed that normetanephrine and corticosterone were reversible, competitive inhibitors and phenoxybenzamine was an irreversible inhibitor of extraneuronal uptake in the smooth muscle cells. 4. Two methods of analysis were used to obtain estimates of the affinities of the reversible inhibitors for extraneuronal uptake. The Ki values calculated by Marquardt non-linear least squares regression analysis and Hunter-Downs method, respectively, were normetanephrine: 2.75 microM and 3.47 microM, and corticosterone: 1.52 microM and 1.44 microM. 5. Normetanephrine and corticosterone had higher affinities than adrenaline for extraneuronal uptake in the trachealis smooth muscle cells.

Kinetics of the O-methylating system for isoprenaline in the trachea and aorta of rabbit.

Segments of tracheal smooth muscle or aorta from rabbits pretreated with reserpine (1 mg/kg) were incubated in 3H-isoprenaline (0.5-60 mumol/l). Steady-state rates of O-methylation were determined by measuring the formation of 3-O-methylisoprenaline (OMI) after incubation of tracheal and aortic tissues for 30 min and 10 min, respectively. The steady-state O-methylation of isoprenaline in rabbit trachea was saturable, at least up to 60 mumol/l isoprenaline. In rabbit aorta, the O-methylation appeared to be saturable up to 30 mumol/l isoprenaline, but the rate of O-methylation increased for higher concentrations. The Km values for the saturable component of O-methylation were 11.8 mumol/l in trachea and 3.03 mumol/l in aorta. The Vmax values were 0.51 nmol X g-1 X min-1 in trachea and 0.56 nmol X g-1 X min-1 in aorta. In tissues incubated in 0.5 mumol/l isoprenaline, 100 mumol/l corticosterone caused 78% inhibition of OMI formation in trachea and 86% inhibition in aorta. There was no inhibition of OMI formation by 100 mumol/l corticosterone in tracheal or aortic tissues incubated in 60 mumol/l isoprenaline. Model calculations showed that the experimental results in trachea and aorta (3. above) were consistent with (a) entry of isoprenaline into the cells in the tissues by extraneuronal uptake and diffusion, and (b) exposure of the isoprenaline to intracellular catechol-O-methyltransferase with Vmax enzyme much less than Vmax uptake.

The contribution of diffusion to the entry of catecholamines into guinea-pig trachealis smooth muscle cells.

In smooth muscle or cartilage preparations of guinea-pig trachea incubated in 0.1 mumol/l 3H-isoprenaline, extraneuronal uptake inhibitors (corticosterone, normetanephrine, phenoxybenzamine) caused only partial inhibition (64-75%) of the formation of 3-O-methylisoprenaline. Thus, isoprenaline appeared to be exposed to catechol-O-methyltransferase by diffusional entry as well as by extraneuronal uptake in both the smooth muscle and cartilage regions of the trachea. Fluorescence microphotometric measurements of catecholamine uptake in trachealis smooth muscle cells, when tissues were incubated in 200 or 1,200 mumol/l isoprenaline, adrenaline or noradrenaline in the absence and presence of extraneuronal uptake inhibitors (corticosterone, normetanephrine, phenoxybenzamine), showed that the contribution of diffusional entry to the uptake of the amines into the cells fitted with the order of their lipophilicities, viz. isoprenaline much greater than adrenaline greater than noradrenaline. A kinetic analysis of the uptake of isoprenaline into the trachealis smooth muscle cells was carried out in the absence and presence of 100 mumol/l corticosterone. The kinetic analysis (a) showed that the corticosterone-resistant component of total uptake was not saturable, supporting the view that it represented diffusional entry of isoprenaline into the cells, and (b) provided Km and Vmax values (112 mumol/l and 101 F/min, respectively) for the saturable extraneuronal uptake of isoprenaline into the cells. The study provided evidence for marked diffusional entry of the lipophilic amine isoprenaline into guinea-pig trachealis smooth muscle cells. The diffusional entry of adrenaline was much less and that of noradrenaline negligible.(ABSTRACT TRUNCATED AT 250 WORDS)

The extraneuronal compartments for the distribution of isoprenaline in the rat heart.

The distribution of 3H-isoprenaline in the perfused rat heart was re-examined. After initial loading with 3H-isoprenaline hearts were washed out with amine-free solution; the efflux curves were subjected to the peeling technique, and half times for efflux and compartment sizes were determined. In contrast to earlier reports from his department (Bönisch et al. 1974;l Bönisch 1978), 3H-isoprenaline was found to distribute mainly into one extra-neuronal compartment, irrespective of whether COMT was intact or inhibited (by the presence of U-0521). It was also not influenced by pretreatment of the animals with reserpine. This type of distribution was influenced neither by the concentration of isoprenaline nor by the duration of the loading of the tissue with the amine. The one major extra-neuronal distribution compartment of 3H-isoprenaline has the characteristics of the "old" compartment III: it has a relatively short half time for the efflux of 3H-isoprenaline and it has a high activity of COMT. Moreover, corticosterone inhibits the inward and outward flux of 3H-isoprenaline into and from compartment III. The Ki for the inhibition by corticosterone of the efflux of 3H-isoprenaline (2 mumol/l) is very similar to the Ki for impairment of uptake2 (determined by Bönisch 1978). Apart from the major distribution compartment III, two minor distribution compartments were detected: On the one hand, experiments with hearts which had an intact COMT revealed that a minor distribution compartment IV (characterized by a long half time for efflux and by an absence of COMT activity) may exist, although its magnitude does not exceed one tenth of the former compartment IV. In addition, part of the quickly equilibrating (and rather small) compartment II was corticosterone-sensitive. When the results of Azevedo et al. (1983 are considered together with the present results, compartment III appears to represent the uptake of 3H-isoprenaline into myocardial cells, while it is likely that radioactivity accumulated in the smooth muscle of blood vessels may constitute the corticosterone-sensitive part of compartment II.

The effect of various ions on uptake2 of catecholamines.

The effects of a decrease of the K+ gradient on the extraneuronal inward transport and outward movement of catecholamines were studied in rat heart, rabbit aortic rings and guinea-pig trachealis smooth muscle. Elevation of the extracellular K+ concentration caused a) inhibition of the corticosteroid-sensitive extraneuronal uptake (uptake2) of 3H-isoprenaline in rat heart and of 3H-noradrenaline in rabbit aorta, and b) acceleration of efflux of 3H-isoprenaline from rat heart, 3H-noradrenaline from rabbit aorta and adrenaline (measured by microphotometry) from guinea-pig trachealis muscle. In rat heart and rabbit aorta, the acute omission of one or the other of the ions Na+, Cl-, K+ or Ca2+ from the perfusion of incubation medium had no effect on initial rates of uptake2 of catecholamines, except that the absence of K+ had a small inhibitory effect in the rat heart. The prolonged absence of Na+, Ca2+ or K+ from the perfusion or incubation medium caused a marked inhibition of uptake2 of catecholamines. These inhibitory effects developed more quickly in rat heart than in rabbit aorta. These results are compatible with the possibility that either the K+ gradient across the cell membrane or the resting membrane potential is the force driving uptake2.

Analysis of the O-methylated metabolites of isoprenaline, adrenaline and noradrenaline in physiological salt solutions by high-performance liquid chromatography with electrochemical detection.

This study provides the first report of a sensitive, simple and rapid high-performance liquid chromatographic (HPLC) assay for the simultaneous analysis of isoprenaline and its metabolite, 3-O-methylisoprenaline, in samples of physiological salt solutions. The assay does not require time-consuming sample clean-up or extraction procedures and uses a Nova-Pak C18 column, an isocratic mobile phase and an amperometric detector. In addition, small modifications to the composition of the mobile phase have also provided sensitive assays for noradrenaline and adrenaline and their O-methylated or O-methylated deaminated metabolites (normetanephrine, metanephrine, 3-methoxy-4-hydroxyphenylethylene glycol and 3-methoxy-4-hydroxymandelic acid). These HPLC assays are sufficiently sensitive and rapid to replace the use of [3H]amines and column chromatographic separation of the metabolites for most in vitro studies on the uptake and subsequent metabolism of catecholamines by monoamine oxidase and/or catechol-O-methyltransferase in tissues.

A study designed to explore the hypothesis that beta-1 adrenoceptors are "innervated" receptors and beta-2 adrenoceptors are "hormonal" receptors.

It has been demonstrated that the chronotropic responses to sympathomimetic amines in rat atria were mediated only by beta-1 adrenoceptors. This was like guinea pig (beta-1) but unlike cat (beta-1 and beta-2). Extraneuronal uptake of isoproterenol into rat atrial myocardial cells was detected by fluorescence histochemistry but uptake was less than was seen in cat atria. Furthermore, it did not modulate responses of rat atrial preparations to isoproterenol. The lack of specific extraneuronal uptake in guinea-pig atrial myocardial cells was confirmed. Collation of data on the atria of these three species with data already available on guinea-pig trachea allowed the following conclusions. All four tissues contained a population of beta-1 adrenoceptors and were adrenergically innervated. Beta-2 adrenoceptors were present in cat atria and guinea-pig trachea and these tissues possessed a functionally effective extraneuronal metabolizing system for catecholamines. Beta-2 adrenoceptors were not detected in guinea-pig or rat atria and in these tissues extraneuronal uptake was either absent (guinea pig) or not functionally effective (rat). It is suggested that these data could support the hypotheses that 1) beta-1 adrenoceptors are "innervated" receptors mediating responses to neuronally released norepinephrine, whereas beta-2 adrenoceptors are "hormonal" receptors mediating responses to circulating epinephrine and 2) extraneuronal metabolizing systems are of particular importance in the dissipation of circulating catecholamines acting on beta-2 adrenoceptors.