(1968) showed that high K+ evokes secretion of noradrenaline (NA) from the sympathetic nerves of the cat spleen in a calciumdependent manner. It has also been reported that agents which increase the duration of the action potential, such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP), markedly potentiate the release evoked by nerve stimulation (Thoenen, Haefely & Staehelin, 1967;Gillespie & Tilmisany, 1976;Wakade, 1980) Wakade (1981aWakade ( , 1982 further extended these observations in the guinea-pig heart, showing that TEA (20 mM) enhanced the response to 35 and 70 mM K+ i) The Macmillan Press Ltd 1983 278 S.M. KIRPEKAR, J.C. PRAT & M.T. SCHIAVONE by 5 fold and 1.5 fold, respectively, and that TTX blocked this potentiation. They suggested that a part of the release induced by high K+ is due to regenerative activity, since the response was partially blocked by TTX and potentiated by TEA.The primary purpose of this study was to reassess the secretory response to excess K+ in the cat spleen. Our previous investigations of K+-evoked NA release in the cat spleen focused on responses to very high K+ concentrations (bolus injection of 3.7 M K+ or prolonged application of 140 mM K+) (Kirpekar & Wakade, 1968;Kirpekar et al., 1976; Kirpekar etal., 1977;Garcia, Kirpekar & Pascual, 1978). In the present study, we have used intermediate concentrations of K+ to re-examine the role of regenerative depolarization in K+-evoked secretion and the mechanisms of muscarinic inhibition and aautoinhibition in the splenic nerve terminals.
Methods
Spleen slicesCats were anaesthetized with ether. The abdomen was opened by a midline incision, then the spleen was quickly removed and cut into transverse sections (0.5 to 0.7 mm thick), using a tissue slicer. To label the NA stores of the splenic nerve, the slices were incubated for 30 min with 10 ml of Krebs solution containing 10 pCi of (±)-[3H]-NA (specific activity 7.5 Ci/mmol), then washed 3 times in 20 ml of Krebs solution over a 30 min period. In each experiment, a number of slices (approximately 100 mg) were incubated in 10 ml of Krebs solution for 5 min to determine the background release, then transferred to 10 ml of a test solution for 5 min. The incubation temperature was 37°C.
Perfused adrenal glandLeft and right adrenal glands were prepared for retrograde perfusion at room temperature according to the procedure of Garcia, Hernandez, Horga & Sanchez-Garcia (1980). The perfusion rate was about 1 ml/min. Glands were perfused for about 30 min before the start of each experiment.
Perfusion and incubation solutionsKrebs bicarbonate buffer consisted of (mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4-7H2O 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 10. When excess K+ (as K2SO4) was added, an osmotic equivalent of NaCl was concomitantly removed. For studies with La3", 2+or 10mMNa, a Tris-buffered solution was used, consisting of (mM): NaCl 143, KCl 6.7, CaC12 2.5, MgSO4-7H20, 2.4, Tris (hydroxymethyl)-aminomethane (Tris) 5.0, and glucose 10. When NaCl was decreased to 10 mM, 266 mM su...