Modulation of noradrenaline release in human cardiovascular tissues.
- Manfred Göthert
- Pharmacology & toxicology
The antagonistic potency, pA2, of several non-selective i3-antagonists on presynaptic (3-adrenoceptors was evaluated using a parallel line assay and MacKay's equation against isoproterenol-induced increases in 3H release in isolated guinea-pig pulmonary arteries preloaded with 3H-norepinephrine. Cumulatively applied isoproterenol at 10-9 M, 10-8 M and 10-7 M dose-dependently increased 3H release evoked by transmural field stimulation at 1 Hz . 19-Antagonists tested dose-dependently antagonized the isoproterenol-induced increases. The order of pA2 was carteolol (11.23+0.09)>nadolol (9.78±0.05)>pindolol (9.59±0.03) >propranolol (9.26±0.17). Carteolol has the highest pA2 and is a useful tool for clarifying whether or not presynaptic (3-adrenoceptors tonically function. It has been proposed that there is a positive feedback mechanism of the release of the transmitter norepinephrine through presy naptic (3-adrenoceptors (1-3). For example, isoproterenol augments contractile responses of human saphenous vein to electrical field stimulation with small relaxation during con traction elicited by exogenously applied norepinephrine (4), whereas propranolol alone stereoselectively inhibits contractile responses of guinea-pig pulmonary arteries to sympathetic nerve stimulation without modifications of contraction induced by exogenously applied norepinephrine (5). Classical pharmacological techniques to determine the pA2 values have been widely used for the evaluation of the antagonistic activities of various antagonists on different types of postsynaptic receptors (6). However, the kinetic analysis of the properties of presynaptic (3-adrenoceptors has not been done (2, 3), although the subtype of these adrenoceptors was qualitatively determined as the (32 type in the peripheral sympathetic nerves (2, 3, 5, 7). There is a possibility that presynaptic (3-adrenoceptors might differ from postsynaptic (3-adrenoceptors, probably in a similar manner as the dominant pre synaptic and postsynaptic a-adrenoceptors differ in their affinity for agonists and for antagonists (1). In general, there is extreme controversy or inconsistency concerning the blocking actions of (3-antagonists alone on presynaptic (3-adrenoceptors (2, 3). A potent 13-antagonist on presynaptic 13-adrenoceptors should be found to clarify whether or not these receptors tonically function in the positive feedback mechanism. Thus, a trial to estimate a pA2 value has been done for a quantitative analysis of antagonistic activities of several non-selective (3-antagonists against isoproterenol-induced increases in impulse evoked release of 3H from guinea-pig pulmonary arteries preloaded with 3H norepinephrine. Spirally cut preparations of the pulmonary arteries from male guinea-pigs, weighing 200 to 250 g, were prepared and incubated at 37'C for 60 min with oxygenated Krebs bicarbonate solution containing 10-7 M 3H norepinephrine (Amersham/Searle) and ascorbic acid 100 mg/I as described pre viously (5, 7). After rinsing for 10 min with norepinephrine-free medium, the preparations were mounted vertically between a pair of platinum stimulating electrodes and super fused with Krebs' solution at a constant rate of 1 ml/min. The solution had the following composition (mM): NaCl, 120.7; KCI, 5.9; CaC12, 2.5; MgC12, 1.2; NaHC03, 15.5; NaH2PO4, 1.2; and glucose, 11.5. The solution, maintained at 37'C, pH 7.2 to 7.4, was bubbled with 5% C02 in 02. A 90 min equilibration period was allowed and then transmural field stimulations (1 Hz, 2 msec, 10 V, 100 pulses) were repeated 4 times (S, to S4 periods) at 15 min intervals, using an electrical stimulator with an isolator (SEN 3201 and SS-120J, Nihon Kohden). Super fusate for 2 min was collected before, during and after the nerve stimulation, respectively, and 12 ml of ACS-II solution was added, and total 3H activities expressed as disintegration per min were determined using a liquid scintillation spectrometer (Packard 2660). Impulse-evoked release of 3H was calculated as the difference between resting efflux before stimulation and total efflux detected in 3 successive samples during and after stimulation. /-Isoproterenol at 10-9 M, 10-8 M and 10-7 M was cumulatively applied 6 min after the S1, S2 and S3 period of stimulation, respec tively, in the absence or presence of each 3 to 4 dose of 8-antagonists. (3-Antagonists were applied 25 min before S, and were present throughout the experiments. The effect of each dose of isoproterenol was evaluated by the % release ratio of S2/S,, S3/S, and S4/S,, respectively. According to the method of parallel line assay (8), the parallelism between the 2 dose-response curves of isoproterenol, one in the absence and the other in the presence of a given dose, [I], of a 4-antagonist, was ascertained to be undeniable, the distance between 2 regression lines with a common regression coefficient was statistica ly calculated, and then a dose ratio (DR) was determined from the distance. A pA2 value at the given dose of the (3 antagonist was calculated from MacKay's equation (9): pA2=log (DR-1)-log [I]. Log (DR-1) was plotted against a log molar dose (6), a regression line was drawn by the method of least squares, and the slope and its S. E. were calculated (10). Drugs used were /-isoproterenol hydro chloride (Sigma), d/-carteolol hydrochloride (Otsuka), nadolol (Squibb Dalnippon), pin dolol (Sandoz-Sankyo) and /-propranolol hydrochloride (ICI). Pindolol was dissolved in 0.1 N HCI, and further dilution was made with Krebs' solution. The other drugs were dissolved in distilled water. The resting 3H efflux and the 3H efflux evoked by transmural field stimulation at 1 Hz from spirally cut pulmonary arteries preloaded with 3H-norepinephrine were 3286.9±670.1 dpm/tissue/2 min and 5380.3±302.6 dpm/tissue/100 pulses (n=7) 90 min after the start of superfusion. The evoked release ratios of S2/S,, S3/S, and Table 1. Effects of cumulatively applied Isoproterenol at 10-9 M, 10-8 M and 10-7 M on impulse-evoked 3H release from spirally cut guinea -pig pulmonary arteries preloaded with 3H-norepinephrine and carteolol