SUMMARY1. Cat spleens were perfused with Krebs bicarbonate solution using a constant flow pump. The amount of noradrenaline released during splenic nerve stimulation was measured at various frequencies. The dependence of noradrenaline release on the ionic composition of perfusion medium was also determined.2. The effect of frequency of stimulation on the output of noradrenaline was studied in both normal and phenoxybenzamine treated cats. In normal cats, the output was 0 33 ng/stimulus at 10/sec, whereas it was 1-21 ng/ stimulus at 30/sec. In phenoxybenzamine-treated cats, the maximum output of noradrenaline of 4 ng/stimulus was obtained at 5 or 10/sec. Higher or lower frequencies of stimulation produced lower output.3. In both normal and phenoxybenzamine treated cats, removal of calcium from the perfusing medium nearly abolished the release of noradrenaline in response to nerve stimulation. Replacement of calcium restored the noradrenaline release. The noradrenaline output/stimulus was linearly related to the log of the external calcium concentration.4. Increasing the concentration of magnesium to (10-20 mM) reduced the noradrenaline output. This depressant effect of magnesium was partially antagonized by increasing the calcium concentration of the perfusion solution.5. Divalent alkali metal earths such as barium and strontium were able to substitute for calcium. Barium substitution nearly doubled the noradrenaline output/stimulus and increased the pressor activity of the samples taken just before nerve stimulation.6. Removal of potassium from the perfusion fluid or lowering the sodium concentration to 50 mm had little effect on the release of noradrenaline. Lowering the sodium concentration to 37X5 mm or less usually abolished the noradrenaline output; this effect is attributed to blockade of nerve conduction. 220 S. M. KIRPEKAR AND Y. MISU 7. It is suggested that depolarization of post-ganglionic sympathetic nerve terminals may increase the influx of calcium ions which in turn leads to the release of noradrenaline from the nerve terminals.

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Sympathetic nerve stimulation and intralummal norepinephrine infusion for more than 15 seconds produced a biphasic response in the isolated rabbit ear artery perfused with RREBS solution. This response consisted of an initial rapid constriction (phase A), which was followed by partial relaxation, and a final slowly developing constriction (phase B), which lasted for the duration of nerve stimulation or norepinephrine administration. Raising the potassium concentration of the Krebs solution to 12mM decreased the relaxation time between the two constrictor phases in response to norepinephrine; lowering the potassium concentration to 1.2 mM increased the relaxation lime and decreased the degree of constriction of both phases. Biphasic vasoconstrictor responses were also elicited by the intraluminal infusion of phenylephrine, histamine, serotonin, or 35 BM potassium. When calcium was absent from the perfusing solution or when manganous sulfate (LMW) was present, norepinephrine produced only a fast phase A constriction, with no subsequent slow phase B constriction. However, after treatment of the trtery with ryanodine, the phase A constriction in response to norepinephrine was markedly inhibited, but the phase B constriction was not. We concluded that the fast phase A constriction depends on the release of calcium from an intracellular pool and that the slow phase B constriction depends on the influx of extracellular calcium.

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SUMMARY1. Cat spleens were perfused with Krebs-bicarbonate solution by means of a constant flow pump. The amount of noradrenaline released by an injection of potassium chloride solution (3.7M) was measured. The dependence of noradrenaline released by KCI on the ionic composition of perfusion medium was determined.2. In normal cats, the average output was 166 ng following a low dose of KCI (0'2 ml.), and 507 ng following a high dose (0.8 ml.). After phenoxybenzamine treatment, the outputs with both doses were nearly doubled.3. In both normal and phenoxybenzamine treated cats, removal of calcium from the perfusing solution nearly abolished the release of noradrenaline. Replacing the calcium in Krebs solution restored the output. Increasing the magnesium concentration to 20 mm also markedly reduced the noradrenaline output.4. Lowering the sodium concentration to either 25 or 0 mm increased the noradrenaline output by nearly two-to threefold. In solutions with reduced concentrations of sodium, noradrenaline was released over longer periods following KCl injections. In phenoxybenzamine treated cats, reduction of sodium in the perfusing solution also increased the noradrenaline output by nearly twofold following either the low or the high dose of KCI.5. It is concluded that calcium ions are necessary for the release of noradrenaline from post-ganglionic sympathetic nerves following depolarization by potassium ions. It is also suggested that sodium ions are specifically needed for the reincorporation of released transmitter into the sympathetic nerve endings.

SUMMARY1. 4-aminopyridine (4-AP, 1 mM) increased noradrenaline (NA) output from the perfused cat spleen at 5 Hz by about fivefold. Enhancement of NA release by 4-AP was reversible. Output of NA induced by potassium was not affected.2. NA output was doubled at low concentrations (0.1-0.3 mM) of 4-AP, but maximal effect was obtained at 1-3 m . At 10 m , it induced spontaneous release of NA which was insensitive to calcium. 4. 4-AP at pH 8-5 was more effective in enhancing NA release than at pH 7-4. 5. 4-AP increased the recovery of intra-arterially infused NA from the control 26 to 47 %.6. 4-AP did not affect release of catecholamines (CA) from the perfused cat adrenal gland by acetylcholine (ACh).7. It is suggested that 4-AP inactivates potassium current in sympathetic nerves and prolongs the duration of the action potential, thereby allowing a greater influx of calcium ions into the neurone to enhance release of NA.

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3. Calcium did not cause spontaneous release of noradrenaline either when high concentrations were injected directly into the spleen or after first perfusing the spleen with calcium-free medium.4. Carbachol, protoveratrine and high potassium inhibit, whereas TEA, barium and rubidium enhance, the evoked release of noradrenaline.5. The relation of noradrenaline release to influx of calcium ions and its modification by various agents has been discussed.