Effect of hypothermia on beta 1-adrenoceptor-mediated relaxation of pig bronchus.

The relaxant potencies of (+/-)-isoprenaline and of (-)-noradrenaline (NA) in the pig isolated bronchus were increased 5.4 and 3.1 fold respectively by lowering the organ bath temperature from 37 degrees C to 27 degrees C, whereas the potencies of the non-catecholamine beta-adrenoceptor agonists fenoterol and orciprenaline were not significantly changed. At 37 degrees C, the catechol-O-methyl transferase (COMT) inhibitor U-0521 (30 microM), caused a 7.2 fold increase in the potency of isoprenaline but had no effect on the potency of fenoterol. At 27 degrees C the potency of isoprenaline was similar in the absence or presence of U-0521 (30 microM). Furthermore, in bronchi where extraneuronal uptake was inhibited by phenoxybenzamine, the potency of NA was not significantly altered by reducing bathing temperature from 37 degrees C to 27 degrees C. These results suggest that the hypothermic potentiation of isoprenaline in pig bronchus resulted from inhibition of COMT or of access to COMT, rather than from sensitization of beta 1-adrenoceptors.     

Metabolism of [3H]-(+/-)-isoprenaline by isolated atria and coronary arteries of the kitten

1 Isolated coronary arteries of the kitten accumulated more unchanged isoprenaline and metabolized more amine than atria following incubation for 1 to 20 min with [3H]-(+/-)-isoprenaline (25 ng/ml or 5 micrograms/ml). 2 Cortisol (10 or 80 microM), U-0521 (120 microM) and oxytetracycline (100 microM) all reduced metabolite formation. 3 Cortisol inhibited 'Iso InfluxMin' (cellular isoprenaline accumulation plus total metabolite production). In contrast, it increased, decreased or did not alter accumulation of unmetabolized isoprenaline, depending upon the experimental conditions. 4 Isoprenaline accumulation was increased in atria and reduced in coronary arteries by U-0521, while oxytetracycline reduced accumulation in coronary arteries at the high amine concentration. 5 It is concluded that in atria, cortisol inhibits metabolism and has differential effects on a number of extraneuronal compartments which accumulate isoprenaline. Both cortisol and U-0521 appear to be extraneuronal uptake inhibitors and inhibitors of catechol-O-methyltransferase in coronary arteries. Oxytetracycline may have effects additional to inhibition of isoprenaline binding to connective tissue fibres.     

Effect of steroids on beta-adrenoceptor-mediated relaxation of pig bronchus.

1--Progesterone, testosterone (40 microM), cortisol and cortisol hemisuccinate (80 microM) caused 6-8 fold potentiations of (+/-)-isoprenaline (Iso)-induced relaxations of pig bronchus while several other steroids caused smaller potentiations or had no effect. 2--17 beta-Oestradiol (40 microM) increased the potency of Iso, (-)-adrenaline (Adr) and (-)-noradrenaline (NA) by 10.6, 2.3 and 2.6 fold respectively but had no significant effect on the potency of fenoterol (Fen). 3--Inhibition of catechol-O-methyl transferase (COMT) with U-0521 (30 microM) caused a 6 fold increase in the potency of Iso but failed to alter the potency of Adr, NA or Fen. The extraneuronal uptake inhibitor normetanephrine (50 microM) caused significant 2 fold increases in the potency of Iso and Adr but did not potentiate the responses to NA or Fen. 4--In preparations where the potency of Iso had already been increased by U-0521 (30 microM) or by normetanephrine, 17 beta-oestradiol produced no significant further increase in potency. These results indicate that steroid-induced increases in the potency of catecholamines in pig bronchus can be explained in terms of inhibition of COMT or extraneuronal uptake or both.     

Further study of the adrenoceptors of the saphenous vein of the dog: Influence of factors which interfere with the concentrations of agonists at the receptor level

In the present study the affinities of some sympathomimetic amines for α- and β-adrenoceptors of the dog saphenous vein tissue were determined after all known factors interfering with the concentration of these agonists at the receptor level had been assessed and excluded. It was observed that in control experiments the relative potencies of sympathomimetic agonists for inducing contractions were: adrenaline (1.6) > noradrenaline (1.0) > phenylephrine (0.38) > isoprenaline (0.009).The elimination of neuronal uptake by cocaine, 4 × 10^?6M, enhanced predominantly the effects of noradrenaline (by a factor of 7.5), whereas block of catechol-O-methyl transferase (COMT) by U-0521, 10^?4 M, only enhanced those of adrenaline (by a factor of 2.6) and block of β-adrenoceptors by propranolol, 5 × 10^?7 M, enhanced those of isoprenaline (by a factor of 3) and adrenaline (by a factor of 1.8).Block of COMT enhanced the effects of adrenaline approximately as much as did the blockade of neuronal uptake; this seems to indicate that the affinity of adrenaline for extraneuronal and neuronal uptake processes is approximately the same.Regarding the relaxation-inducing capacity of sympathomimetic agents it was observed that isoprenaline, adrenaline and noradrenaline are full agonists, whereas phenylephrine was not able to produce relaxation amounting to more than 5% of the maximum. Denervation did not modify the relaxant effects of isoprenaline. After elimination of all known factors interfering with the concentration of the sympathomimetic agonists in the biophase, the ratios between the ED₅₀'s of each amine for α- and β-adrenoceptors were: adrenaline = 34, noradrenaline = 109 and isoprenaline = 0.0041.     

The guinea-pig trachea O-methylating system is more effective in modulating beta 2- than beta 1-adrenoceptor-mediated responses to isoprenaline

Assuming that responses of the guinea-pig trachea to isoprenaline in the presence of atenolol (10 mumol L-1) are exclusively, or at least predominantly, beta 2-adrenoceptor mediated and that responses to isoprenaline in the presence of ICI 118,551 (erythro-DL-1(7-methylindan-4-yloxyl)-3-isopropylaminobut an-2-ol) (1 nmol L-1) are exclusively, or at least predominantly beta 1-adrenoceptor mediated, the influence of inhibition of COMT by U-0521 (dehydroxy-2-methyl propiophenone) (50 mumol L-1) has been compared in both conditions. U-0521 enhanced beta 2-adrenoceptor mediated responses to isoprenaline 3.3-fold, while those mediated by beta 1-adrenoceptors were enhanced only 2.2-fold. It is concluded that in guinea-pig trachea COMT activity is functionally more effective in modulating responses which are mediated by beta 2-adrenoceptors than responses mediated by beta 1-adrenoceptors.     

Kinetic constants of isoprenaline and corticosterone for extraneuronal uptake in different cell types from various tissues

Extraneuronal uptake of isoprenaline was studied in a number of different, histologically identified, cell types: trachealis smooth muscle cells, vascular smooth muscle cells and myocardial cells. Segments of cat, rat or rabbit trachea, dog coronary artery or cat atria were incubated in isoprenaline, in the presence of U-0521 (100 mumol l-1) to inhibit catechol-O-methyltransferase. The intensities of formaldehyde-induced fluorescence (due to accumulation of isoprenaline) were measured, using microphotometry, in the appropriate cells in histological sections of the tissue. Endogenous fluorescence in adrenergic nerves was removed by pretreatment of rats with 6-hydroxydopamine and of cats and rabbits with reserpine. Segments of dog coronary artery were incubated with 6-hydroxydopamine in vitro to remove neuronal fluorescence. This treatment was also shown to be effective in guinea-pig trachea without any influence on the determination of the kinetic parameters of isoprenaline uptake in the trachealis smooth muscle cells. Fluorescence in all cell types studied was shown to represent isoprenaline which had accumulated by extraneuronal uptake, in that fluorescence was reduced by drugs which inhibit extraneuronal uptake (corticosterone, normetanephrine, metanephrine and/or phenoxybenzamine), by exposure to a K+-Krebs solution and by incubating tissues in isoprenaline at 0 degree C rather than 37 degrees C. In each cell type, isoprenaline uptake obeyed Michaelis-Menten saturation kinetics, both in the absence and presence of corticosterone. Corticosterone was a competitive inhibitor of isoprenaline uptake in all the cell types.(ABSTRACT TRUNCATED AT 250 WORDS)     

The neuronal and extraneuronal uptake and metabolism of 3H-(−)-noradrenaline in the perfused rat heart

1. Hearts were obtained from reserpine-pretreated rats and perfused with 0.95 micron 3H(-)-noradrenaline. The rate of removal of 3H-noradrenaline from the perfusion fluid was measured (from the arterio-venous difference) as well as the rate at which the 3H-metabolites appeared in the venous effluent. 2. When either 30micron corticosterone was added under steady-state conditions during perfusion with 3H-noradrenaline (to inhibit neuronal and extraneuronal uptake, respectively), each inhibitor reduced the removal of noradrenaline by about 50%; in the presence of both inhibitors removal was abolished. 3. Dihydroxymandelic acid (DOMA) was of neuronal, normetanephrine (NMN) of extraneuronal origin; dihydroxyphenylglycol (DOPEG) and the OMDA fraction (containing methoxyhydroxyphenylglycol-MOPEG-and methoxyhydroxymandelic acid-VMA) were formed both neuronally and extra-neuronally. 4. The extraneuronal metabolism of 3H-noradrenaline was in quick equilibrium with the 3H-noradrenaline in the perfusion fluid; most of the total formation of DOPEG, MOPEG and NMN was recovered from the venous effluent. 5. Extraneuronally formed DOPEG, MOPEG and NMN distributed in the tissue with half times corresponding to their half time for efflux. 6. Inhibition of monoamine oxidase (MAO) by pargyline increased the extraneuronal formation of NMN; MAO and catechol-O-methyl transferase (COMT) appear to be contained in the same extraneuronal compartment. 7. The extraneuronal accumulation of 3H-noradrenaline required 30 min or more to reach a steady state; inhibition of one or both enzymes slowed this process. Inhibition of MAO increased the extra-neuronal accumulation of 3H-noradrenaline; inhibition of COMT failed to do so, since the enzyme inhibitor (U-0521) was a weak inhibitor of extra-neuronal uptake. 8. The rate constants for the efflux of the metabolites of noradrenaline decreased in the order of MOPEG greater than DOPEG greater than NMN greater than DOMA greater than VMA.     

Comparison of the uptake and O-methylation of isoprenaline by cardiac and respiratory tissues of guinea-pigs

An attempt was made to correlate the extraneuronal uptake and O-methylation of isoprenaline in cardiac and respiratory tissues with the predominant beta-adrenoceptor subtype known to mediate the sympathetic responses of these tissues. Papillary muscles, left atria, trachealis muscles and lung parenchymal strips of guinea-pigs were incubated with [3H]isoprenaline ([3H]ISO) (0.1 microM) for 60 min. Levels of total radioactivity and of separated [3H]ISO and [3H]O-methylisoprenaline ([3H]OMI) in each tissue and of [3H]OMI in the medium were determined. U-0521 (10(-4) M) inhibited tissue O-methylation and caused an elevation of unchanged [3H]ISO in the tissues. The latter effect was attributed to the fact that the normal conversion of [3H]ISO to [3H]OMI did not occur. Metanephrine (10(-5) and 10(-4) M) did not affect tissue levels of unchanged [3H]ISO, but reduced tissue levels of [3H]OMI. Levels of [3H]OMI in tissue and medium were only slightly reduced, indicating possible extracellular sites of O-methylation. In the presence of U-0521, metanephrine (10(-4) M) reduced the accumulation of [3H]ISO, indicating that metanephrine was an inhibitor of the extraneuronal uptake of isoprenaline. It is concluded that two cellular compartments for O-methylation exist, access to one being dependent upon metanephrine-sensitive extraneuronal uptake. The extraneuronal uptake capacities of the tissues (when O-methylation was inhibited) was in the order papillary muscle less than lung = atria less than trachea. Cellular O-methylating capabilities, measured from tissue [3H]OMI, was in the order papillary muscle less than atria = trachea less than lung. These orders are discussed in relation to the beta-adrenoceptor mediating the response of each tissue and to the reported degree of sympathetic innervation.     

Relation between the amount of smooth muscle of venous tissue and the degree of supersensitivity to isoprenaline caused by inhibition of catechol-O-methyl transferase

Summary The relation between the smooth muscle cell mass of dog saphenous vein strips and the degree of supersensitivity to isoprenaline caused by U-0521 (3,4-dihydroxy-2-methyl propiophenone), an inhibitor of the catechol-O-methyl transferase (COMT), was studied. For the quantitative determination of smooth muscle mass, the thickness of the muscle layer as determined by light microscopy and the maximal shortening induced by supramaximal concentration of phenylephrine were used.After the strips had been contracted by 3×10^–6 M phenylephrine, a concentration which was able to produce an about 90% maximal contraction, dose-response curves to the relaxant effect of isoprenaline were determined in the absence and in the presence of U-0521 (10^–4 M).It was observed that U-0521 caused marked supersensitivity to the relaxant effect of isoprenaline (varying between 3 and 81 times), as well as an increase of the maximal relaxation caused by this amine (varying between 7 and 120%). The correlation between these data and the smooth muscle cell mass shows that there was a direct proportionality between these parameters.Oxytetracycline (10^–4 M), an inhibitor of binding of catecholamines to collagen, did not produce any enhancement of the effects of isoprenaline.It is concluded that COMT is related to smooth muscle cells in this tissue.This study was supported by a grant from Instituto de Alta Cultura (PMC-2).Preliminary results of this study were presented to the Joint Meeting of German, Hungarian, Portuguese and Yugoslav Pharmacological Societies (Graz, 2–5 September, 1974).     

Age-related changes in guinea pig respiratory tissues: considerations for assessment of bronchodilators

Tracheal tissues from immature guinea pigs were significantly more sensitive to histamine and carbachol than their counterparts from mature animals. Bronchial but not tracheal tissues from immature animals were more sensitive to isoproterenol and salbutamol than tissues from mature animals. Functional antagonism between carbachol-induced tension and salbutamol-induced relaxation was greatest in bronchial tissues from immature animals. Functional antagonism was not evident when a concentration of histamine required to induce maximal but not supramaximal tension was used to induce tone. Inhibition of catechol-o-methyl transferase (COMT) with U 0521 (10 microM) or inhibition of extraneuronal uptake with hydrocortisone (200 microM), in tissues from animals in either age group, affected neither the potency of, nor the extent of relaxation induced by, isoproternol or salbutamol in histamine-contracted tissues. We conclude that extraneuronal uptake and metabolism by COMT do not modify tissue sensitivity to the relaxants studied. Therefore age-related changes in the relaxant properties of isoproterenol and salbutamol are not due to differences in their uptake or metabolism but probably reflect changes at the receptor level.     

The handling of five catecholamines by the extraneuronal O-methylating system of the rat heart

In a comparative study, the handling of five catecholamines by the extraneuronal O-methylating system of the rat heart was determined; all rats were pretreated with reserpine, monoamine oxidase and neuronal uptake were inhibited in all experiments. Hearts were perfused for 7 min with a tracer concentration of 3H-(+/-)-isoprenaline, either in the absence or in the presence of unlabelled catecholamines (which reduced the O-methylation of the tracer amine). IC50's were determined for unlabelled catecholamines and then converted to "half-saturating outside concentrations", i.e., to those concentrations in the perfusion fluid that half-saturate the intracellular catechol-O-methyl transferase (COMT). The values for the (-)-isomers of dobutamine, isoprenaline, adrenaline and noradrenaline and that for dopamine were low and rather similar (between 0.67 and 2.7 mumol/l). Stereoselectivity for isoprenaline probably reflected the preference of uptake2 for the (-)-isomer. The effects of (-)- and (+)-dobutamine indicated that both isomers are a) transported by uptake2 and b) good substrates of COMT. The Vmax for O-methylation [determined for 3H-(+/-)-isoprenaline, 3H-(+/-)-adrenaline, 3H-(+/-)-noradrenaline and 3H-dopamine] was rather similar for all four catecholamines. It is concluded that the extraneuronal O-methylating system of the rat heart handles the five catecholamines in a similar manner, although the Km for uptake2 had been found to increase substantially in the order: dobutamine less than isoprenaline less than adrenaline less than noradrenaline less than dopamine (Grohmann and Trendelenburg 1984b).     

The fate of isoprenaline in the isolated rabbit aorta

Summary The fate of isoprenaline (ISO) was studied in the intact rabbit aorta, as well as in the isolated adventitia and isolated media, by means of liquid scintillation counting of ³H-ISO and ³H-O-methylisoprenaline (OMI) as well as by autoradiography of tissues incubated with 2M ISO. The 3 preparations accumulated and O-methylated ISO; O-methylation was proportionally higher in the intact vessel than in its constituents. The isolated adventitia differed from the other preparations in reaching a steady-state of cortexone-resistant accumulation after 8 min of incubation and in exhibiting an O-methylating capacity which was partly resistant to the COMT inhibitor, U-0521. The effect of cocaine gave evidence for a participation of neuronal uptake in the accumulation of the amine in the intact aorta. In the media, most of the accumulation occurred in smooth muscle cells and was reduced by cortexone and increased by U-0521; elastin and collagen, present both in the media and the adventitia, showed accumulation which was not affected by the inhibitory drugs studied.The results show that in the rabbit aorta there is a corticosteroid-resistant uptake mechanism (which predominates in the adventitia) which involves structures devoid of COMT activity. Furthermore, smooth muscle cells represent, in the media, the extraneuronal metabolizing site of loss. The adventitia is a complex layer, with different types of cells which may intervene in accumulation and metabolism of ISO. Therefore, it is concluded that the isolated media represents an acceptable model for the study of both corticosteroidsensitive and-resistant extraneuronal mechanisms, whereas the isolated adventitia is characterized by the presence of neuronal and extraneuronal mechanisms.Some of the results were presented to the 3rd Meeting on Adrenergic Mechanisms, Porto, July 12–13, 1978 (Proceedings, p. 93) and to the 1st Joint Meeting of the Spanish and Portuguese Pharmacological Societies (Santiago de Compostela, 21–23.5.79). Supported by Instituto Nacional de Investigação Científica (Centro de Farmacologia e Biopatologia Quimica-FmPl)     

Effects of extraneuronal uptake inhibitors on the positive chronotropic response to isoprenaline and on the accumulation of isoprenaline in perfused rat heart after inhibition of catechol-O-methyl transferase

Experiments were carried out in isolated perfused rat hearts. The presence of tropolone (100 mumol/l), an inhibitor of catechol-O-methyl transferase (COMT), significantly potentiated the positive chronotropic response to isoprenaline (0.1, 1, 3 and 10 nmol/l). Two uptake2 inhibitors, 3-O-methylisoprenaline (100 mumol/l) and normetanephrine (100 mumol/l), induced a positive chronotropic response, but corticosterone (100 mumol/l) and hydrocortisone (100 mumol/l) had no such effect. 3-O-methylisoprenaline (100 mumol/l) and normetanephrine (100 mumol/l) enhanced the positive chronotropic response to isoprenaline (0.1, 1, 3 and 10 nmol/l). Corticosterone (100 mumol/l) potentiated the positive chronotropic response to isoprenaline (0.1 and 1 nmol/l). Hydrocortisone (30 mumol/l) enhanced the response to 0.1 nmol/l isoprenaline but did not affect the positive chronotropic responses to 1, 3 or 10 nmol/l isoprenaline. The addition of uptake2 inhibitors (3-O-methylisoprenaline, 100 mumol/l; normetanephrine, 100 mumol/l; corticosterone, 100 mumol/l) to the perfusion medium significantly reduced the positive chronotropic response to the perfusion with isoprenaline (3 nmol/l) and tropolone (100 mumol/l). The accumulation of 3H-isoprenaline in the heart perfused with 3H-isoprenaline (1, 10 and 100 nmol/l) was significantly increased by the presence of tropolone (100 mumol/l): the accumulation for 1, 10 and 100 nmol/l of 3H-isoprenaline was 5.07, 47.0 and 500 pmol/g, respectively. The high accumulation observed during perfusion with 3H-isoprenaline (3 nmol/l) and tropolone (100 mumol/l) was significantly decreased by the addition of an uptake2 inhibitor, 3-O-methylisoprenaline (100 mumol/l), normetanephrine (100 mumol/l) or corticosterone (100 mumol/l), but not by hydrocortisone (30 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Catechol-O-methyltransferase inhibition by U-0521 increases striatal utilization of levodopa

Summary The effects of U-0521, a catechol-O-methyltransferase (COMT) inhibitor, were studied on this enzyme activity and on Dopa metabolism in rat striatum. In vivo maximal inhibition (95%) of COMT activity was obtained at 5 min with enzyme recovery to 64% of basal activity at 120 min. When injected in increasing doses U-0521 (200 mg·kg^–1) inhibited, at 10 min, COMT activity by 85% with an IC₅₀=80 mg·kg^–1. In rats pretreated with U-0521 and then with DOPA the accumulation of 3-O-methyldopa (OMD) in the plasma was essentially blocked while Dopa, dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) accumulation in the striatum was significantly higher than in DOPA treated controls. U-0521, a potent COMT inhibitor, enhances the availability and utilization of levodopa in the brain and may thus be helpful in future treatment of parkinsonian patients.     

Temperature-induced changes in dissociation constants (KA) of agonists at cardiac beta-adrenoceptors determined by use of the irreversible antagonist Ro 03-7894.

The positive inotropic responses of guinea-pig left atria and papillary muscles and positive chronotropic responses of right atria to sympathomimetic amines were examined at 38 degrees and 30 degrees C. At the lower temperature, supersensitivity to orciprenaline and isoprenaline was exhibited as shifts of the dose-response curves to the left and significant reductions in EC50 values. This supersensitivity could not be attributed to reduced metabolism since the experiments were performed in the presence of metanephrine (10(-5)M) and U-0521 (3',4'-dihydroxy-2-methylpropiophenone) (10(-4)M) as inhibitors of extraneuronal uptake and catechol-O-methyltransferase (COMT) respectively, and the agonists are not susceptible to neuronal uptake. After incubation of the tissues with Ro 03-7894 (1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol), followed by its prolonged washout (greater than 2h), the maximum responses to isoprenaline and orciprenaline were depressed, confirming the apparently irreversible beta-adrenoceptor antagonism. Dissociation constants (KA) for isoprenaline and orciprenaline were determined from the equiactive concentrations obtained before (A) and after (A') incubation with Ro 03-7894, plotted as 1/A against 1/A' (KA = (slope-1)/intercept). KA values were the same for orciprenaline in the three cardiac preparations and for isoprenaline in the atria. This applied at 38 degrees and 30 degrees C and indicates that the beta-adrenoceptors mediating the inotropic and chronotropic responses of the guinea-pig heart do not differ. The KA values of both agonists were, however, consistently and significantly lower at 30 degrees than at 38 degrees C, indicating an increase in affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of noradrenaline and isoprenaline removal in the canine hindlimb and kidney

Because isoprenaline is not a substrate for neuronal uptake (Uptake-1, U-1), the difference in regional removal of isoprenaline from regional removal of the sympathetic neurotransmitter noradrenaline has been proposed as an index of regional U-1 activity. U-1 activity has not been assessed in the kidney, where decreased U-1 may account for increased renal spillover of noradrenaline into plasma in disorders such as essential hypertension. Tracer-labelled noradrenaline and isoprenaline were simultaneously infused intravenously into anaesthetized dogs, and the regional removal of noradrenaline and isoprenaline was measured in the hindlimb and kidney after administration of the U-1 blocker desipramine, hydrocortisone, which inhibits extra-neuronal uptake of noradrenaline (Uptake-2, U-2), or no drug. Hindlimb removal of noradrenaline (51%) exceeded that of isoprenaline (36%). Desipramine abolished this difference by decreasing removal of noradrenaline without affecting removal of isoprenaline. Renal removal of isoprenaline exceeded that of noradrenaline (74% vs 54%) even after U-1 blockade. Hydrocortisone did not affect removal of noradrenaline or isoprenaline in either bed. The results suggest that differences in removal of noradrenaline and isoprenaline reflect U-1 activity in the hindlimb but not in the kidney; U-1 is much more important than U-2 in the regional removal of noradrenaline; and one mechanism of noradrenaline removal in the kidney is by neuronal uptake.     

The influence of block of catechol-O-methyl transferase on the sensitivity of isolated organs to catecholamines

Summary The sensitivity of various isolated organs to catecholamines was tested before and after block of catechol-O-methyl transferase (COMT) by U-0521 (3,4-dihydroxy-2-methyl propiophenone; 18 g/ml for 20 min). Isolated Nictitating Membrane of the Cat. The sensitivity to catecholamines was increased by inhibition of COMT whenever the experimental conditions resulted in a high potency of the amine, i.e., when the ED50 of the amine (in the absence of U-0521) was below about 10^–6 M. Thus, block of COMT potentiated the effects of (–)-noradrenaline and (–)-adrenaline after denervation (or in the presence of cocaine) but not on muscles with an intact adrenergic innervation; it also increased the sensitivity to the-effects but not to the-effects of isoprenaline. No potentiation was observed for dopamine which has a low potency even after denervation. The relation between potency of the catecholamine and the degree of the sensitizing effect of U-0521 was not due to saturation of COMT or of access to the enzyme. Measurements in denervated muscles of the production of O-methylated metabolites from (±)-noradrenaline-H³ (added to the bath for 20 min in concentrations of about 10^–7 to 10^–4 M), and of the extraneuronal accumulation of noradrenaline-H³ did not reveal any saturation of either the enzyme or the extraneuronal accumulation of the amine.When block of COMT resulted in supersensitivity to a catecholamine, the time required to reach steady-state responses was usually increased. This is consistent with the view that block of the enzyme impaired a site of loss.Block of COMT failed to produce any substantial potentiation of the effects of the indirectly acting amines, tyramine and mephentermine. O-methylation of the released transmitter seems to occur after the amine has reached the receptors of the effector cells. Isolated Strips of Cat and Bat Spleen. Results were quantitatively similar, since block of COMT potentiated the effects of (–)-noradrenaline and (–)-adrenaline in the presence but not (or only very little) in the absence of cocaine. However, the degree of supersensitivity after block of COMT (in the presence of cocaine) was smaller than in the nictitating membrane. In the cat spleen, cocaine causes a small but significant degree of supersensitivity to isoprenaline but not to methoxamine. Isolated Sat Aorta, Block of COMT did not increase the effect of cocaine.It is suggested that differences in the morphology of the adrenergic innervation contribute to the observed organ differences.With the technical assistance of Miss Roneen D. Hobbs.This study was supported partly by Research Grant NB-07131 from the National Institutes of Health, U.S. Public Health Service, and partly by the Deutsche Forschungsgemeinschaft.     

The influence of oestrogen and oestrogen metabolites on the sensitivity of the isolated rabbit aorta to catecholamines

Summary 1. In the present study the influence of oestradiol, catechol oestrogens, and O-methylated oestrogens was determined on the contractile responses of the isolated rabbit aorta to (–)-adrenaline. 2. Oestradiol (40 mol/l), 2-hydroxyoestradiol (2OHE₂) (20 mol/l), and 2-methoxyoestradiol (2MeOE₂) (20 mol/l) all sensitized the rabbit aorta to contractile responses to (–)-adrenaline. Similarly, the 2-hydroxy and 2-methoxy derivatives of oestrone and oestriol also sensitized the aorta to (–)-adrenaline-induced contractions. The largest degree of sensitization was seen in the presence of the 2-methoxysteroids. 3. Oestradiol and 2OHE₂ did not increase responses of the aorta to (–)noradrenaline, while slight potentiation of contraction was seen in the presence of 2MeOE₃. 4. The potentiating effect of 2OHE₂ on contractile responses to (–)-adrenaline was abolished by prior treatment of the tissue with a COMT inhibitor (U-0521, 55 mol/l). Conversely, pretreatment of the tissue with 2OHE₂ prevented the augmented aortic contraction to (–)-adrenaline usually seen after inhibition of COMT. The non-additive nature of the sensitization seen after combined treatment with 2OHE₂ and U-0521 was qualitatively similar to that seen following combined exposure to maximally effective concentrations of U-0521 and an inhibitor of extraneuronal uptake (hydrocortisone 100 mol/l). 5. Oestradiol and 2MeOE₂ reduced the formation of both the ³H-O-methylated, ³H-deaminated and the ³H-O-methylated deaminated metabolites of ³H-(–)-adrenaline (0.15 mol/l) during exposure of the aorta to the tritiated catecholamine. Treatment of the aorta with the extraneuronal uptake inhibitor phenoxybenzamine (33 mol/l) produced a similar inhibition of formation of all ³H-metabolites of ³H-(–)-adrenaline. 6. While preincubation with 2OHE₂ markedly reduced the formation of ³H-O-methylated and ³H-O-methylated deaminated metabolites from ³H-(–)-adrenaline (0.15 ol/l), the formation of ³H-deaminated adrenaline metabolites was considerably enhanced by exposure to 2OHE₂. Pretreatment of the aorta with the COMT inhibitor U-0521 produced a similar effect on ³H-adrenaline metabolism.7. Oestradiol, 2OHE₂, and 2MeOE₂ did not alter the tritium contents retained in segments of aorta incubated with ³H (±)-isoprenaline (0.15 ol/l). In contrast, the three steroids reversed the enhanced retention of tritium in the aorta induced by prior inhibition of COMT (U-0521 55 mol/l).8. It is concluded that the biotransformation of oestradiol to its 2-hydroxy and 2-methoxy metabolites does not diminish the potentiating actions of the steroids on adrenaline-induced aortic contractions. Oestradiol and 2MeOE₂ appear to produce their effects through inhibition of the extraneuronal amine uptake process, while 2OHE₂, a substrate for COMT, probably exerts is sensitizing through a combination of inhibition of the extraneuronal uptake process and competitive inhibition of COMT.Abbreviations 2OHE₁ 2-hydroxyoestrone - 2OHE₂ 2-hydroxyoestradiol - 2OHE₃ 2-hydroxyoestriol - 2MeOE₁ 2-methoxyoestrone - 2MeOE₂ 2-methoxyoestradiol - 2MeOE₃ 2-methoxyoestriol - U-0521 34-dihydroxy-2-methylpropiophenone Supported by the part in West Virginia University Biomedical Fund; American Heart Association, West Virginia Affiliate; and NIH grants HL30351 and K04-HL01228Selected aspects of this study were presented to the American Society for Pharmacology and Experimental Therapeutics (Barone et al. 1985; Panek et al. 1986) Send offprint requests to R. E. Stitzel at the above address